Porous planar cell capture system and method of use

ABSTRACT

The invention relates to a porous planar cell capture system for use in determining the presence and/or amount of cells, for example, viable cells, in a liquid sample, and to methods of using such a cell capture system. The cell capture system contains a fluid permeable, planar membrane adopted to retain cells thereon, a fluid permeable support member that supports the membrane, and an optional register associated with the membrane.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of and claims the benefit of priorityto co-pending U.S. patent application Ser. No. 13/875,969, filed May 2,2013, which claims priority to and the benefit of U.S. ProvisionalPatent Application No. 61/641,812, filed May 2, 2012, and U.S.Provisional Patent Application No. 61/784,807, filed on Mar. 14, 2013;the entire contents of each application are incorporated herein byreference.

FIELD OF THE INVENTION

This invention relates generally to cell capture system for use indetermining the presence and/or amount of cells, for example, viablecells, in a liquid sample, and to methods of using such a cell capturesystem.

BACKGROUND

Microbial contamination by, for example, Gram positive bacteria, Gramnegative bacteria, and fungi, for example, yeasts and molds, may causesevere illness and, in some cases, even death in human and animalsubjects. Manufacturers in certain industries, for example, food, water,cosmetic, pharmaceutical, and medical device industries, must meetexacting standards to verify that their products do not contain levelsof microbial contaminants that would otherwise compromise the health ofa consumer or recipient. These industries require frequent, accurate,and sensitive testing for the presence of microbial contaminants to meetcertain standards, for example, standards imposed by the United StatesFood and Drug Administration or Environmental Protection Agency.

Depending upon the situation, the ability to distinguish between viableand non-viable cells can also be important. For example, during themanufacture of pharmaceuticals and biologics, it is important that thewater used in the manufacturing process is sterile and free ofcontaminants. Furthermore, it is important that water contained inmedicines (for example, liquid pharmaceutical and biological dosageforms, for example, injectable dosage forms) and liquids (for example,saline) that are administered to a subject, for example, vianon-parenteral routes, is also sterile and free of contaminants. On theother hand, the presence of some viable microorganisms in drinking watermay be acceptable up to a point. In order to be potable, drinking watermust meet exacting standards. Even though microorganisms may be presentin the water supply the water may still be acceptable for humanconsumption. However, once the cell count exceeds a threshold level, thewater may no longer be considered safe for human consumption.Furthermore, the presence of certain predetermined levels ofmicroorganisms in certain food products (for example, fresh produce) anddrinks (for example, milk) may be acceptable. However, once those levelshave been exceeded the food or drink may be considered to have spoiledand no longer be safe of human consumption.

Traditional cell culture methods for assessing the presence of microbialcontamination and/or the extent of microbial contamination can takeseveral days to perform, which can depend upon the organisms that arebeing tested for. During this period, the products in question (forexample, the food, drink, or medical products) may be quarantined untilthe results are available and the product can be released. As a result,there is a need for systems and methods for rapidly detecting (forexample, within hours or less) the presence and/or amount of microbialcontaminants, in particular, viable microbial contaminants, in a sample.

SUMMARY

The invention is based, in part, upon the discovery of a cell capturesystem that can be used to determine the presence of viable cells in acell containing sample. The cell capture system can be used incombination with an optical detection system that detects the presenceof viable cells in the sample. The results can be used to measure thebioburden (for example, to measure the number and/or percentage and/orfraction of viable cells) of a particular sample of interest.

In one aspect, the invention provides a cell capture system. The systemcomprises a fluid permeable, planar membrane comprising an exposed firstsurface, at least a portion of which is adapted to retain cells thereon.The portion (i) defines a plurality of pores having an average diameterless than about 1 μm so as to permit fluid to traverse the portion ofthe membrane while retaining cells thereon, (ii) is substantiallynon-autofluorescent when exposed to light having a wavelength in a rangefrom about 350 nm to about 1000 nm, and (iii) has a flatness toleranceof up to about 100 μm. The cell capture system optionally furthercomprises a register (for example, line, spot, or other feature)associated with the membrane so as to permit the determination of thelocation of cells retained on at least a portion of the planar membrane.

The membrane can be of any of a variety of shapes, for example,circular, annular, ovoid, square, rectangular, elliptical, etc., and canhave some portion or all of one side exposed for cell retention.Moreover, the membrane may form one or more apertures therein toaccommodate a mask and may be formed from several separate membranesassembled together with the mask or other structural element. In oneembodiment, the membrane may be in the shape of a disc, for example, asubstantially planar disc. The membrane (for example, in the form of adisc) can have a thickness in a range selected from the group consistingof from 1 μm to 3,000 μm, from 10 μm to 2,000 μm, and from 100 μm to1,000 μm.

In certain embodiments, the cell capture system further comprises afluid permeable support member adjacent at least a portion of a secondsurface of the membrane that is opposite the first surface of themembrane. The fluid permeable support, for example, in the form of arigid porous plastic frit, retains enough fluid to maintain moisture inthe porous membrane disposed adjacent the permeable support, which incertain embodiments, can help maintain the viability of cells retainedon the porous membrane. The support member can have a thickness in arange selected from the group consisting of from 0.1 mm to 10 mm, from0.5 mm to 5 mm, and from 1 mm to 3 mm.

In certain embodiments, the cell capture system further comprises a maskproximate at least another portion of the first surface of the membrane.Depending upon the design configuration (for example, when the porousmembrane is a disk), the mask can be circular.

In certain embodiments, the cell capture system further comprises aplurality of detectable particles, for example, fluorescent particles.The fluorescent particles can be adapted to be excited by a beam oflight having a wavelength at least in a range from about 350 nm to about1000 nm, or a wavelength in a range from about 350 nm to about 600 nm ora wavelength in a range from about 600 nm to about 750 nm. The particlescan be used as part of a positive control to ensure that one or more ofthe cell capture system, the cell capture method, the detection system,and the method of detecting the viable cells are operating correctly.

Depending upon the design of the cell capture system, the particles (forexample, fluorescent particles) can be pre-disposed upon at least aportion of the porous membrane or disposed within a well formed in amask. Alternatively, the particles (for example, fluorescent particles)can be mixed with the liquid sample prior to passing the sample throughthe porous membrane. In such an approach, the fluorescent particles canbe dried in a vessel that the sample of interest is added to.Thereafter, the particles can be resuspended and/or dispersed within theliquid sample. Alternatively, the fluorescent particles can be presentin a second solution that is mixed with the liquid sample of interest.Thereafter, the particles can be dispersed within the liquid sample.

In certain embodiments, the cell capture system, in particular theporous membrane, has a sterility assurance level less than 10⁻⁶, 10⁻⁷,10⁻⁸, or 10⁻⁹. This can be achieved, for example, by sterilizing thecell capture system, via techniques known in the art, for example,autoclaving, exposure to ionizing radiation, for example, gammaradiation, or exposure to a sterilizing fluid or gas, for example,ethylene oxide. The cell capture system can be enclosed within areceptacle, for example, a bag, prior to, during, or aftersterilization. It is contemplated that the cell capture system can beplaced within a receptacle, for example, within a bag that is sealed,for example, hermetically sealed, before terminal sterilization, forexample, via exposure to ionizing radiation.

In another embodiment, the invention provides a cell capture cupcomprising an open cylindrical portion and an annular seal adapted tomate and engage with a base comprising the cell capture system of anyone of the foregoing aspects and embodiments. The cell capture cup andthe base can have a sterility assurance level less than 10⁻⁶, 10⁻⁷,10⁻⁸, or 10⁻⁹, which can be achieved using any of the approachesdiscussed hereinabove.

In another aspect, the invention provides a method of determining thepresence and/or amount of cells in a liquid sample. The method comprisesthe steps of: (a) capturing cells present in the sample on the any oneof the cell capture system and/or the cell capture cup disclosed herein,and (b) determining the presence or amount of cells captured in step(a). The method can further comprise the step of labeling the capturedcells with a detectable moiety, for example, a fluorescent label. Thedetermining step can utilize an optical detector, for example, afluorescence detector.

In another aspect, the invention provides a method of detecting presenceof viable cells, and/or measuring the viability of cells, in a liquidsample. The method comprises the steps of: (a) capturing cells presentin the sample and/or the cell capture cup disclosed herein; (b)selectively labeling captured viable cells; and (c) detecting thepresence of cells labeled in step (b) and/or measuring the viability ofcells labeled in step (b). The cells can be labeled using at least oneof a viability stain and a viability staining system, each of which cancomprise a fluorescent moiety. The labeled viable cells can be detectedwith an optical detector, for example, a fluorescence detector.

BRIEF DESCRIPTION OF THE DRAWINGS

In the drawings, like reference characters generally refer to the sameparts throughout the different views. Also, the drawings are notnecessarily to scale, emphasis instead generally being placed uponillustrating the principles of the invention. In the followingdescription, various embodiments of the present invention are describedwith reference to the following drawings, in which:

FIG. 1A is a schematic representation of an exemplary detection systemthat can be used to determine the presence and/or amount of viable cellsin a cell sample;

FIG. 1B is a schematic perspective view of an exemplary detection systemwith a door in a closed position;

FIG. 1C is a schematic perspective view of the exemplary detectionsystem of FIG. 1B with the door in an open position;

FIG. 1D is a schematic perspective view of the exemplary detectionsystem of FIG. 1B with a touchscreen in a raised position;

FIG. 2A is a schematic top view of an exemplary membrane assembly;

FIG. 2B is a schematic, exploded side view of the membrane assembly ofFIG. 2A.

FIGS. 3A and 3B are schematic representations of exemplary membraneassemblies;

FIG. 4A is a schematic, exploded perspective view of an exemplarymembrane assembly having a permeable membrane and a fluid permeablesupport member;

FIG. 4B is a schematic side view of the exemplary permeable membraneassembly of FIG. 4A;

FIG. 5A is a schematic perspective view of an exemplary cell capture cupand a corresponding base;

FIG. 5B is a schematic partial cut-away view of the cup and base of FIG.5A showing a membrane assembly;

FIG. 5C is a schematic perspective view of the cup, base, and membraneassembly of FIG. 5B in an unassembled state;

FIG. 5D is a schematic perspective view of the base of FIG. 5A;

FIG. 5E is a schematic partial cross-sectional view of the cup and baseof FIG. 5B with a different membrane holder assembly and posts from aseparate holder;

FIGS. 6A-6D are schematic perspective, side, top, and bottom views,respectively, of a cup assembly having a lid, a cup, a membrane, and abase;

FIG. 6E is a schematic bottom perspective view of the lid of FIG. 6A;

FIG. 6F is a schematic side view of the lid of FIG. 6A;

FIGS. 7A-7D are schematic perspective, side, top and bottom views,respectively, of a cup member shown in the cup assembly of FIG. 6A;

FIGS. 8A-8D are schematic perspective, top, bottom, and side views,respectively, of the base of the cup assembly of FIG. 6A;

FIG. 9A is a schematic perspective view of the base of FIG. 8A showing apartial cut away view of the membrane and underlying permeable supportmember;

FIGS. 9B-9D are schematic top, bottom, and side views of the base(complete membrane and underlying permeable support member) of FIG. 9A;

FIG. 10A is a schematic exploded perspective view of the cup assembly ofFIG. 6A;

FIG. 10B is a schematic cross-sectional view of the cup assembly of FIG.6A, without a membrane and a permeable support member;

FIG. 10C is a schematic cross-sectional view of the base of FIG. 9A witha membrane, a permeable support member, and a base lid;

FIG. 11A depicts a process for capturing cells on a permeable membrane;

FIG. 11B depicts a schematic exploded perspective view of a chuck,stage, base, support member, membrane, and base lid components;

FIG. 12A is a schematic representation of the spectral overlap betweenthe emission of an exemplary fluorescent dye (dashed line, in units offluorescence intensity (I_(F))) and the absorption spectrum of anexemplary quencher (solid line, in units of absorbance or opticaldensity (O.D.));

FIGS. 12B-12C are schematic representations showing a partial overlapbetween the emission spectra of an exemplary fluorescent dye and anexemplary quencher demonstrating two approaches for selecting adye-quencher pair for use in the cell viability assays discussed herein;

FIG. 13A is a schematic exploded cross-sectional side view of anexemplary membrane holder for use with the system of FIG. 1A;

FIG. 13B is a schematic of a configuration of magnets for use with themembrane holder (stage) of FIG. 13A;

FIG. 13C is a schematic exploded perspective view of the membrane holder(stage) of FIG. 13A with a membrane assembly and chuck;

FIG. 13D is a schematic perspective view of the membrane holder,membrane assembly, and chuck of FIG. 13C in an assembled configuration;

FIGS. 14A-14C are schematic perspective, top, and bottom views,respectively, of an exemplary stage;

FIGS. 15A-15D are schematic perspective, top, bottom, and side views,respectively, of an exemplary chuck;

FIG. 16A is a schematic perspective view of an exemplary membrane holder(stage) for receiving a base;

FIG. 16B is a schematic perspective view of an exemplary base for usewith the membrane holder of FIG. 16A;

FIG. 16C is a schematic perspective view of the exemplary membraneholder of FIG. 16A and the base of FIG. 16B in an unassembledconfiguration;

FIG. 16D is a schematic perspective view of the exemplary membraneholder of FIG. 16A and the base of FIG. 16B in an assembledconfiguration showing posts extending from the membrane holder andpassing through operatives defined by the base;

FIG. 17A is a schematic representation of viable (live) and non-viable(dead) cells following staining with a membrane permeable fluorescentdye that permeates both viable and non-viable cells and a membraneimpermeable quencher that selectively permeates non-viable cells;

FIG. 17B is a schematic representation of viable (live) and non-viable(dead) cells following staining with a membrane permeable nucleic acidbinding fluorescent dye that permeates both viable and non-viable cellsand a membrane impermeable nucleic acid binding quencher thatselectively permeates non-viable cells;

FIG. 18 is a schematic representation of a region of a permeablemembrane showing viable and non-viable cells stained with an exemplaryviability staining system shown in FIG. 17A or 17B;

FIGS. 19A and 19B are pictorial representations of viable cells andnon-viable cells, respectively, with positive control beads depicted ineach figure;

FIGS. 20A and 20B depict phase contrast and fluorescence images,respectively, for viable and non-viable cells using an exemplaryviability staining system;

FIGS. 21A and 21B are phase contrast and fluorescence images,respectively, for viable and non-viable cells using an exemplaryviability staining system;

FIG. 22 is an image of viable cells (E. coli and Candida albicans)captured on a permeable membrane, stained with an exemplary viabilitystaining system and detected as fluorescent events on a rotating discusing the detection system shown of FIG. 1A;

FIGS. 23A-J are phase contrast and fluorescence images of mixedpopulation of viable and non-viable microorganisms stained withexemplary pairs of nucleic acid binding fluorescent dyes and a nucleicacid binding fluorescence quenchers; and

FIGS. 24A-E are phase contrast and fluorescence images of mixedpopulations of viable and non-viable microorganisms stained withexemplary pairs of nucleic acid binding fluorescent dyes and a nucleicacid binding fluorescence quenchers, where the cells were imaged usingan epifluorescent microscope (FIGS. 24 A-D, where phase contrast imagesare shown in FIGS. 24A and C, and the corresponding fluorescence imagesare shown in FIGS. 24 B and D, respectively) or using a detection systemshown in FIG. 1A.

DESCRIPTION

The instant invention is directed to a cell capture system, a method ofcapturing cells (including viable cells) in the cell capture system, amethod for selectively staining viable cells, (e.g., viable cellscaptured in the cell capture system), and to a method for determiningthe presence and/or amount of viable cells in a cell sample (e.g., aliquid sample). The cell capture system and the various methods can beused, either alone or in combination, to determine the presence and/oramount of viable cells in a cell containing sample and, in particular,can be used to determine the bioburden (e.g., to measure the numberand/or percentage and/or fraction of viable cells in a sample) of aparticular sample of interest. The cell capture system and the variousmethods can be used to measure the bioburden of cells in a liquid sample(e.g., a water sample), a comestible fluid (e.g., wine, beer, milk, babyformula or the like), a body fluid (e.g., blood, lymph, urine,cerebrospinal fluid or the like), growth media, a liquid sample producedby harvesting cells from a source of interest (e.g., via a swab) andthen dispersing and/or suspending the harvested cells, if any, in aliquid (e.g., buffer or growth media).

It is contemplated that, by using the devices and methods describedherein, it will be possible to determine the presence and/or amount ofviable cells in sample within less than approximately 2 hours, less thanapproximately 1 hour, or even less than approximately 30 minutes afterthe cells have been captured on a porous membrane of the cell capturesystem. It is contemplated, however, depending upon the desiredsensitivity, it is possible to culture the cells captured on the porousmembrane (e.g., for 15 minutes to several hours) to permit cellproliferation. Nevertheless, by using the devices and methods describedherein, even when including a culturing step, it is possible todetermine the presence and/or amount of viable cells in a sample muchfaster than other technologies available in the art.

Each of the various aspects and certain embodiments of the inventionwill be discussed in detail below.

(I) Cell Capture System

The cell capture system described herein can be used with an opticaldetection system that detects the presence of viable cells. The resultscan be used to measure the bioburden (e.g., to measure the number and/orpercentage and/or fraction of viable cells in a sample) of a particularsample of interest. Exemplary detection systems are described, forexample, in International Patent Application No. PCT/IB2010/054965,filed Nov. 3, 2010, U.S. patent application Ser. No. 13/034,402, filedFeb. 24, 2011, International Patent Application No. PCT/IB2010/054966,filed Nov. 3, 2010, U.S. patent application Ser. No. 13/034,380, filedFeb. 24, 2011, International Patent Application No. PCT/IB2010/054967,filed Nov. 3, 2010, and U.S. patent application Ser. No. 13/034,515,filed Feb. 24, 2011. One embodiment of an exemplary system 100, as shownschematically in FIG. 1A, comprises a sample assembly 120 comprising (i)a rotating platform 130 upon which a porous membrane having cellsdisposed thereon rotates about a rotation axis 140, and (ii) a movableplatform 150 that translates linearly (see track 160) relative to adetection system 170 that comprises a light source 180 (e.g., a whitelight source or a laser light source (e.g., a near infrared laser)), andat least one detector 190, for example, a fluorescence detector. A beamof light from light source 180 (excitation light) impinges rotatingplatform 130 and the planar membrane disposed thereon, while emissionlight is detected by detector 190. The light source 180 and the detector190 may be arranged at similar angles relative to the platform 130 asthe beam of light will impact and leave the platform 130 atsubstantially the same angle. In certain circumstances, the detectionsystem consists of a single detector that detects a single wavelengthrange (see FIG. 12B) or multiple wavelength ranges. Alternatively, thedetection system consists of multiple detectors, each of which iscapable of detecting a different wavelength range.

FIGS. 1B-1D depict the exemplary cell detector system 100 having anenclosure 110 and a display (e.g., a touchscreen) 112. The enclosure 110is sized to house the rotating platform 130, which may be accessedthrough a door 114 on the enclosure 110. The enclosure 110 may bemanufactured in various shapes and sizes, including in the depictedrectangular prism form that is approximately 10 in.×10 in.×12 in.(l×w×h). Other shapes may be a cube, cylinder, sphere, or other prism,amongst others. While dimensions vary depending on the shape, theenclosure 110 may range in scale from a few inches to several feet, andpossibly lesser or greater, depending on the application. FIG. 1Bdepicts a cell detection system with the door 114 in a closedconfiguration and FIG. 1C depicts the same system with door 114 in anopen configuration to show rotating platform 130. The touchscreen 112provides a user interface for controlling the operation of the system100, and may display information regarding the system's 100 currentoperating parameters. The touchscreen 112 may be adjustable into a moreupright position (as depicted in FIG. 1D) in order to facilitate easieroperation. In certain embodiments, the touchscreen 112 is only activewhen in the upright position. In other embodiments, the touchscreen 112is always active, or only at select times (e.g., when engaged by auser).

It is understood that such detection systems operate optimally when thecells are disposed upon a solid support or otherwise maintained in aplanar orientation with a tight flatness tolerance (e.g., within aflatness tolerance of up to about 100 μm (±50 μm), e.g., up to about 10μm (±5 μm), up to about 20 μm (±10 μm), up to about 30 μm (±15 μm), upto about 40 μm (±20 μm), up to about 50 μm (25 μm), up to about 60 μm(±30 μm), up to about 70 μm (±35 μm), up to about 80 μm (±40 μm), up toabout 90 μm (±45 μm)), so that the cells can be visualized readily by adetection system within a narrow focal plane. If a dynamic focusingsystem is employed, it is contemplated that flatness tolerances greaterthan 100 μm can be tolerated. Accordingly, it can be preferable to use asupport system that maintains the membrane and any captured cells in asubstantially planar orientation and within a suitably tight flatnesstolerance to permit reliable detection. Depending on the detectionsystem and requirements post detection, the support system may beadapted to present and/or maintain planarity of the membrane when dryand/or when wet or moist after cells have been captured on the solidsupport after passing a cell containing solution through the solidsupport via pores disposed within the solid support.

The invention provides a cell capture system comprising a fluidpermeable, planar membrane comprising an exposed first surface, at leasta portion of which is adapted to retain cells thereon. The portion can:(i) define a plurality of pores having an average diameter less thanabout 1 μm so as to permit fluid to traverse the portion of the membranewhile retaining cells thereon; (ii) be substantially nonauto-fluorescent when exposed to light having a wavelength in a rangefrom about 350 nm to about 1000 nm; and (iii) have a flatness toleranceof up to about 100 μm. The cell capture system 100 optionally furthercomprises a register (e.g., line, spot, or other mark, indicia orstructural feature) associated with the membrane so as to permit thedetermination of the location of cells (for example, the viable cells)retained on at least a portion of the planar membrane. For a disc shapedmembrane, polar coordinates (i.e., radial “r” and angular “θ” coordinatelocations) may be suitable.

The membrane can be of any of a variety of shapes, e.g., circular,annular, ovoid, square, rectangular, elliptical, etc., and can have someportion or all of one side exposed for cell retention. Moreover, themembrane may form one or more apertures therein to accommodate a maskand may be formed from several separate membranes assembled togetherwith the mask or other structural element. In one embodiment, themembrane may be in the shape of a disc, e.g., a substantially planardisc. In certain embodiments, the portion of the porous membrane forcapturing cells and/or particles is greater than 400 mm², 500 mm, 600mm², 700 mm², 800 mm², 900 mm² or 1,000 mm². The membrane (e.g., in theform of a disc) can have a thickness in a range selected from the groupconsisting of approximately from 1 μm to 3,000 μm, from 10 μm to 2,000μm, and from 100 μm to 1,000 μm.

In certain embodiments, the cell capture system 100 further comprises afluid permeable support member adjacent at least a portion of a secondopposing surface of the membrane. The fluid permeable support, forexample, in the form of a smooth planar porous plastic frit, retainsenough fluid to maintain moisture in the porous membrane disposedadjacent the permeable support, which in certain embodiments, can beimportant to maintain the viability of cells retained on the porousmembrane. The support member can have a thickness in a range selectedfrom the group consisting of approximately from 0.1 mm to 10 mm, from0.5 mm to 5 mm, and from 1 mm to 3 mm.

In certain embodiments, the cell capture system 100 further comprises amask proximate at least another portion of the first surface of themembrane. Depending upon the design configuration (e.g., when the porousmembrane is a disk), the mask can be circular or annular, optionallywith radial spokes or supports.

The porous membrane defines a plurality of pores having an averagediameter less than about 1 μm so as to permit fluid to traverse themembrane while retaining cells thereon. In certain embodiments, theaverage pore diameter is about or less than about 0.9 μm, 0.8 μm, 0.7μm, 0.6 μm, 0.5 μm, 0.4 μm, 0.3 μm, 0.2 μm, 0.1 μm, or 0.05 μm. Incertain embodiments, the average pore diameter is about 0.2 μm, and inother embodiments the average pore diameter is about 0.4 μm. Suitablemembranes can be fabricated from nylon, nitrocellulose, polycarbonate,polyacrylic acid, poly(methyl methacrylate) (PMMA), polyester,polysulfone, polytetrafluoroethylene (PTFE), polyethylene and aluminumoxide.

In addition, the porous membrane is substantially non-autofluorescentwhen exposed to light having a wavelength in the range from about 350 nmto about 1,000 nm. As used herein with reference to the porous membrane,the term “substantially non-autofluorescent when exposed to a beam oflight having a wavelength in the range from about 350 nm to about 1,000nm” is understood to mean that the porous membrane emits lessfluorescence than a fluorescently labeled cell or a fluorescent particledisposed thereon when illuminated with a beam of light having awavelength, fluence and irradiance sufficient to cause a fluorescenceemission from the cell or particle. It is understood that a user and/ordetector should be able to readily and reliably distinguish afluorescent event resulting from a fluorescent particle or afluorescently labeled cell from background fluorescence emanating fromthe porous membrane. The porous membrane is chosen so that it ispossible to detect or visualize a fluorescent particle or afluorescently labeled cell disposed on such a porous membrane. Incertain embodiments, the fluorescence emitted from a region of a porousmembrane (e.g., a region having approximately the same surface area as acell or cell colony or particle being visualized) illuminated with abeam of light may be no greater than approximately 30% (e.g., less than30%, less than 27.5%, less than 25%, less than 22.5%, less than 20%,less than 17.5%, less than 15%, less than 12.5%, less than 10%, lessthan 7.5%, less than 5%, or less than 2.5%) of the fluorescence emittedfrom a fluorescent particle or a fluorescently labeled cell, whenmeasured under the same conditions, for example, using a beam of lightwith the same wavelength, fluence and/or irradiance.

Suitable membranes that are non-autofluorescent can be fabricated from amembrane, e.g., a nylon, nitrocellulose, polycarbonate, polyacrylicacid, poly(methyl methacrylate) (PMMA), polyester, polysulfone,polytetrafluoroethylene (PTFE), or polyethylene membrane impregnatedwith carbon black or sputtered with an inert metal such as but notlimited to gold, tin or titanium. Membranes that have the appropriatepore size which are substantially non-autofluorescent include, forexample, ISOPORE™ membranes (Merck Millipore), NUCLEOPORE™ Track-Etchedmembranes (Whatman), ipBLACK Track Etched Membranes (distributed by ARBrown, Pittsburgh, Pa.), and Polycarbonate (PCTE) membrane (Sterlitech).

In order to facilitate accurate detection and count estimation of thecaptured cells, it is beneficial (even essential in some instances,depending on the configuration and capabilities of the detection system)that the membrane is substantially planar (e.g., substantially wrinklefree) during cell detection. As used herein, the term “substantiallyplanar” is understood to mean that an article has a flatness toleranceof less than approximately 100 μm. This is because height imperfections(e.g., wrinkles) may interfere with the optical detection/measurementsystem, leading to erroneous results. As a result, it can be importantfor the porous membrane when dry and/or wet and depending on detectionconditions), retains a relatively tight flatness tolerance, within thedetection capability of the detection system. Various approachesdescribed below allow the porous membrane to be held substantially flatafter cells from a sample fluid are captured thereon and otherapproaches may be apparent to those skilled in the art based on thediscussion herein.

In certain embodiments, the cell capture system further comprises aplurality of detectable particles, for example, fluorescent particles.The fluorescent particles can be adapted to be excited by a beam oflight having a wavelength at least in a range from about 350 nm to about1000 nm, a wavelength in a range from about 350 nm to about 600 nm awavelength in a range from about 600 nm to about 750 nm, or any of thewavelength ranges discussed above. The particles can be used as part ofa positive control to ensure that one or more of the cell capturesystem, the cell capture method, the detection system, and the method ofdetecting the viable cells are operating correctly.

Depending upon the design of the cell capture system, the particles (forexample, fluorescent particles) can be pre-disposed upon at least aportion of the porous membrane or disposed within a well formed in amask. Alternatively, the particles (for example, fluorescent particles)can be mixed with the liquid sample prior to passing the sample throughthe porous membrane. In such an approach, the fluorescent particles canbe dried in a vessel that the sample of interest is added to.Thereafter, the particles can be resuspended and/or dispersed within theliquid sample. Alternatively, the fluorescent particles can be presentin a second solution that is mixed with the liquid sample of interest.Thereafter, the particles can be dispersed within the liquid sample.

FIG. 2A shows an exemplary membrane assembly 200 comprising a porousplanar membrane 202 and a frame (or mask) 204 to hold porous membrane202 substantially flat, i.e., without allowing the formation ofsignificant wrinkles therein. As shown, frame 204 comprises a centralportion 204 a connected to a circumferential portion or outer rim 204 bvia a plurality of spokes (e.g., tensioning spokes) 204 c. One of thespokes denoted 204 c′ may be thicker than the other spokes 204 c andrepresents a register from which the co-ordinates of cells disposed onthe membrane can be measured (for example, r, θ values), where r is theradial distance measured from the axis of rotation and θ is the includedangle between (i) a radial line traversing the point of rotation and thecell and (ii) the register 204 c′.

Membrane 202 comprises a plurality of pores having an average diameterabout or less than about 1 μm, for example, about or less than about 0.9μm, 0.8 μm, 0.7 μm, 0.6 μm, 0.5 μm, 0.4 μm, 0.3 μm, 0.2 μm, 0.1 μm, or0.05 μm. As such, when a liquid fluid containing cells and/or particlescontacts membrane 202, the fluid can traverse through the membrane viathe pores, while the cells and/or particles are retained on a surface ofthe membrane 202. The membrane 202 is substantially non auto-fluorescentwhen exposed to light having a wavelength in the range from about 350 nmto about 1000 nm. Moreover, the membrane 202 has a smooth surface havinga flatness tolerance no greater than about 100 μm when restrained orconfigured for detection by the associated detection system.

As shown in FIG. 2B, membrane 202 has a first surface 214 and a secondsurface 216 that is opposite the first surface. First surface 214 may beaffixed to the frame 204, e.g., via an adhesive bonding layer 218. Thecentral portion 204 a can be affixed to a central portion of membrane202. In the embodiment shown, the diameter of the membrane 202 is aboutthe same as that of the outer rim 204 b, and as a result, the outer rim204 b is affixed to the perimeter of the membrane 202. The spokes 204 cextend radially from the central portion 204 a and may be affixed to themembrane 202. This configuration can hold the membrane 202 substantiallyflat, preventing or minimizing the formation of wrinkles. Furthermore,the formation of wrinkles can also be mitigated or eliminated byapplying downward pressure on the central portion 204 a, which increasesthe surface tension in membrane 202.

In another approach, as depicted in FIG. 3A, a circular membraneassembly 300 comprises a porous membrane 202 having an upper surface304. A circular mask 306, affixed to a central portion of the surface304, holds the membrane 202 substantially flat. In the membrane assembly300, cells in the fluid sample, if any are present therein, are capturedon the exposed portion of the surface 304 that is not covered by themask 306. Membrane assembly 300 may be disposed on a fluid permeableporous support member, as described below, that may maintain the desiredflatness of the membrane during detection. Alternatively oradditionally, in order to keep the membrane 202 substantially flat,downward pressure may be applied to the mask 306. Materials suitable forthe mask 306 include plastic, polycarbonate, polystyrene, polypropylene,and other materials having water repellant properties.

FIG. 3B depicts a membrane assembly 310 that is similar to the membraneassembly 300 shown in FIG. 3A. A mask 316 is similar to the mask 306,but the mask 316 has a protrusion or nipple 318 that allows a user topick up the assembly 310 (including the membrane 202) with fingers orforceps, and transfer the assembly 310 to another location, e.g., on amembrane holder. The top surface of the mask 316 also defines a well 320that may serve as a register so that the location of a particle or celldetected on the surface 304 of the membrane 202 can be described withreference to the location of the well 320. Alternatively or in addition,control particles to be detected may be initially disposed in the well320. In certain other embodiments, the mask may include either theprotrusion or the well, but not both.

In another approach, as depicted in FIGS. 4A and 4B, the porous membrane202 may be disposed in a membrane assembly 400 to maintain the porousmembrane 202 in a substantially planar configuration without the needfor the frame 204 or the masks 306 or 316, by placing the porousmembrane 202 upon a fluid permeable, solid support member 404. In oneembodiment, when the porous membrane 202 is wetted, surface tensionbetween the membrane 202 and the solid support member 404 conforms thebottom surface of the membrane 202 to an upper mating surface 406 of thesupport member 404. For example, in one embodiment, the support member404 may be a fluid permeable, solid substantially planar element thatkeeps membrane 202 in a substantially planar configuration, for example,when the membrane is wetted. The support member 404 is porous, and theupper mating surface 406 is substantially flat and smooth. In anotherembodiment, the solid support member 404 is coated with a non-toxicadhesive, for example, polyisobutylene, polybutenes, butyl rubber,styrene block copolymers, silicone rubbers, acrylic copolymers, or somecombination thereof. When a downward pressure is applied, for example,from a vacuum, the porous membrane 202 becomes loosely adhered to thesolid support member 404, which results in the porous membraneconforming to the surface 406 of the solid support member 404. Thesupport member 404 is porous, and the upper mating surface 406 issubstantially flat and smooth. For example, in one embodiment, thesurface 406 has a flatness tolerance of up to about 100 μm. The diameterof the support member 404 is approximately the same as that of themembrane 202, and preferably the support member 404 has a substantiallyuniform thickness. The support member can have a thickness in a rangeselected from the group consisting of approximately from 0.1 mm to 10mm, from 0.5 mm to 5 mm, and from 1 mm to 3 mm. Materials suitable formaking the porous support member 404 include plastic, polycarbonate,high density polyethylene (HDPE), glass, and metal. In one embodiment,the support member 404 is fabricated by sintering plastic particles madefrom poly (methyl methacrylate) having a mean diameter of 0.15-0.2 mmheld at a temperature near the melting point of the particles and at apressure sufficient to cause sintering of the particles to fuse themtogether and form a uniform structure.

Although the membrane 202 and the support member 404 are depicted ascircular, this is illustrative only. In other embodiments, the membrane202 and/or the support member 404 may be shaped as a square, arectangle, an oval, etc. In general, the shape and the surface area ofthe support member, if it is used, is selected such that the surface ofthe support member is approximately the same size as or slightly smallerthan the membrane disposed thereon.

The membrane 202 is disposed in contact with the substantially flat,smooth surface 406 of the support member 404 before the sample fluid ispoured onto the membrane 202. The generally flat surface 406 helps keepthe membrane 202 substantially flat after the sample fluid is drained.The fluid permeable solid support 404 can also serve as a reservoir forfluid passed through the membrane 202 and the fluid permeable solidsupport 404, to provide the additional benefit of preventing themembrane 202 and viable cells disposed thereon from drying out duringthe detection process. Drying can be detrimental to the viability of thecells retained on the membrane 202.

With reference to FIGS. 5A-5E, a cup and base assembly 500 having a cup502 and a base 504 is used to facilitate the capture of cells present ina liquid sample on a membrane (e.g., the membrane 202) disposed withinthe base 504. The base 504 has a surface 506 (see, FIG. 5D), an outerwall 508, and a lip 510. The surface 506 defines at least one opening512 and, optionally, circular and radial protrusions or grooves 514 tofacilitate drainage of liquid passed through the membrane 202. The wall508 has a circumferential groove 516 under the lip 510. In certainembodiments (see FIG. 5D), the cup 502 comprises a wall 520 having acircumferential protrusion 522 adapted to mate with the base groove 516to releasably interlock the cup 502 to the base 504. A lip section 524of the wall 520, i.e., the section below the protrusion 522, inclinesinwardly to form a circumferential sealing lip adapted to contact anupper surface of the porous membrane 202. The lip section 524 alsocaptures the porous membrane 202 (and in certain embodiments the frame200 and/or the support member 404) between the cup 520 and the base 504.

More generally, a membrane and any components for holding the membranegenerally flat, such as a holder having spokes (described with referenceto FIGS. 2A and 2B), masks (described with reference to FIGS. 3A and3B), and/or the supporting member (described with reference to FIGS. 4Aand 4B) can be received within the cup and base assembly 500 anddisposed on the surface 506 of the base 504. The cup 502 then isdisposed over the membrane assembly such that the wall protrusion 522fits into the groove 516 of the base 504, as depicted in FIG. 5E. Thisfit helps ensure the proper positioning between the cup 502 and the base504, particularly with respect to the membrane 202 containedtherebetween. The dimensions of the section 524 (e.g., the length, theangle of inclination, etc.) are selected such that the section 524presses against the membrane assembly 400 disposed in the base 504 toprovide a fluidic seal and ensure a flat membrane 202.

FIGS. 6A-6D depict another embodiment of a cup and base assembly 550.The cup and base assembly 550 has a cup 552 and a base 554 that in manyaspects function similarly to the cup 502 and the base 504. The cup andbase assembly 550 may also optionally contain a lid 556 for keeping theinterior of the cup 552 protected from contaminants, both before andafter use. A support member 558 (such as the support 404) is disposed inthe base 554 for supporting the membrane 202 (depicted in FIGS. 9A and9B). In the embodiment depicted, the lid 556 is substantially circularto interfit with cup 552, although any complementary shapes would besuitable. The lid 556 is shown in greater detail in FIGS. 6E and 6F,including ridges 560 that provide a small offset between the top of thecup 552 and a bottom surface of the top of the lid 556.

FIGS. 7A-7D depict the cup 552 in greater detail. The cup 552 includesan upper portion 562 that is substantially hollow and tapers out towardsthe top to provide an increased sectional area into which fluid may bepoured. Further tapering directs the fluid toward a lower section 564that is adapted to be received within the base 554. A vertical segment566 can provide increased stability when the cup 552 is disposed withinthe base 554, from which a lip section 568 (similar to lip section 524)extends at an angle. A further vertical section 570 may also be providedfor contacting the membrane 202.

FIGS. 8A-8D depict the base 554. The base 554 includes an outer wall 572defining an upper portion 574 that may catch extraneous fluid. A lowerportion 576 is adapted to be received within a stage (described indetail below), and may be tapered to provide a tight fight when mountedthereon. An interior wall 578 defines a central recess 580 for receivingthe cup 552, and more particularly the vertical segment 566. A tight fitand overlap between the vertical segment 566 and the interior wall 578help ensure a stable fit while the cup 552 is mounted on the base 554. Aledge 582 for receiving the membrane 202 is located at a bottom of theinterior wall 578, and further defines a recess 584 in the middle toreceive the support member 558. The relationship of the base 554, themembrane 202, and the support member 558 is depicted in FIGS. 9A-9D,along with an optional lid 588 (depicted transparently in FIG. 9A).Openings 586 may be provided in the bottom of the base 554, similar tothe openings 512.

In certain embodiments, the cell capture system, in particular theporous membrane, has a sterility assurance level less than 10⁻⁶, 10⁻⁷,10⁻⁸, or 10⁻⁹. This can be achieved, for example, by sterilizing thecell capture system, via techniques known in the art, for example, viaautoclaving, exposure to ionizing radiation, for example, gammaradiation or exposure to a sterilizing fluid or gas, for example,ethylene oxide or vaporized hydrogen peroxide. The cell capture systemcan be enclosed within a receptacle (e.g., a bag), prior to, during, orafter sterilization. The cell capture system can be placed within areceptacle (e.g., a bag) and sealed (e.g., hermetically sealed) beforeterminal sterilization (e.g., via exposure to ionizing radiation).

In another embodiment, the invention provides a cell capture cupcomprising an open cylindrical portion and an annular seal adapted tomate with a base comprising the cell capture system of any one of theforegoing aspects and embodiments. The cell capture cup and base canhave a sterility assurance level less than 10⁻⁶, 10⁻⁷, 10⁻⁸, or 10⁻⁹,which can be achieved using any or all of the approaches discussedherein.

(II) Cell Capture Method

FIG. 10A depicts the components of an exemplary cup and base assembly550. The porous support member 558 and the membrane 202 are disposed inthe center of the base 554. The cup 552 then is installed on top of themembrane 202, helping to maintain the membrane 202 in a flat position.The lid 556 may be provided on top of the cup 552 to protect theinterior of the cup 552 from being contaminated. FIG. 10B depicts thefitting of the components (without the membrane 202 and the support 558.

During use, a sample fluid is poured into the cup 552. Due to the tapersof the cup 552, the fluid wets the membrane assembly and passes throughthe membrane 202. The fluid typically passes through the membraneassembly (e.g., through the membrane 202, and the porous support member558, if one is used) toward the base 554. Negative pressure, forexample, a vacuum, can be advantageously employed to draw fluid throughthe membrane 202 to the openings 586 (e.g., in the embodiment of FIG.5E, via the grooves 514), and to help keep the membrane substantiallyflat. After the fluid is drawn through the cup and base assembly 550,any particles and/or cells in the fluid that cannot pass throughmembrane 202 are retained on the upper exposed surface of the membrane202. After pouring the fluid into the cup assembly, the cup 552 may beseparated from the base 554, as depicted in FIG. 10C, and a lid 558placed on top of the base 554. The lid 588 may be provided on top of thebase 554 to protect the moistened membrane 202 and support 558 fromcontamination when the base is transferred to the stage 802 (FIG. 11B)or when the base containing membrane 202 is incubated, for example, for15 minutes to 8 hours to permit the captured viable cells toproliferate.

An exemplary flow chart showing the assembly of the cell capture system,the passage of liquid sample through the cell capture system and theassembly of the membrane holder for use in an exemplary opticaldetection system is shown in FIG. 11A.

With reference to FIG. 11A, in step 601, a cup and base assembly 550 isprovided. In step 603, the cup and base assembly 550 is coupled to avacuum system (e.g., a vacuum manifold 606) and a negative pressure isapplied to the underside of the cup and base assembly 550. In step 605,the liquid sample is poured into the cup and base assembly 550, and anycells present in the liquid sample are retained on the upper exposedsurface of the porous membrane 202. This pouring step can occur before,at the same time, or after step 603. It is contemplated that thesubstantially non-autofluorescent membrane permits a flow ratetherethrough of at least 5 or at least 10 mL/cm²/min with a vacuum ofabout 5 Torr or about 10 Torr. The cells can then be stained with aviability stain or a viability staining system, for example, asdiscussed in Section III so that it is possible to selectively detectand distinguish viable cells from non-viable cells. The cells mayoptionally be washed with a physiologically acceptable salt and/orbuffer solution to remove residual non-specifically bound fluorescentdye and/or quencher.

In step 607, the membrane assembly is removed from the cup 552,typically in combination with the base 554, though removal independentfrom the base 554 may be possible. In step 609, the base 554 (andthereby the membrane 202) is disposed on a stage 802. In step 611, thestage 802 is disposed on a chuck 804. The stage 802 and the chuck 804are described in greater detail below. Steps 609 and 611 may beperformed in reverse order or concurrently. The stage 802 and the chuck804 can be located in the exemplary detection system 100 of FIG. 1A atthe start of the process in order to detect any cells (viable and/ornon-viable cells) and/or particles captured on the surface of membrane202. In other embodiments, the stage 802 and/or the chuck 804 may beassembled with the base 554 remote from the detection system 100.

(III) Cell Staining

Once the cells are captured on the permeable membrane, the cells can bestained using a viability stain or a viability staining system so as todetect or otherwise distinguish viable cells from non-viable cells. Theparticular staining protocol will depend upon a variety of factors, suchas, the cells being detected, the stain or staining system beingemployed, and whether the cells are going to be stained and detectedimmediately or whether the cells are going to be cultured for a periodof time, for example, from 30 minutes to several hours, to permit thecells to proliferate so that a plurality of cells rather than a singlecell is detected at a particular locus. Exemplary staining and, wheredesired, culturing protocols are discussed in the following sections.

It is understood that a variety of stains and staining systems can beused to selectively stain viable versus non-viable cells. As usedherein, the term “non-viable cells” is understood to mean cells that arealready dead or cells undergoing cell death. Some approaches are basedon the principle that viable cells exclude certain reagents, such astrypan blue, alcian blue, eosin Y, nigrosin, propidium iodide, ethidiumbromide, and ethidium monoazide. For example, when using trypan blue,the non-viable cells stain blue whereas the viable cells are notstained. Other approaches are based upon the principle that viable cellstake up and/or modify certain reagents (for example, fluorescent dyes),where non-viable cells do not. Dyes that selectively label either viablecells or non-viable cells are described in U.S. Pat. No. 5,534,416 andPCT Publication No. WO92/02632, which include esterase-dependent dyes,nucleic acid binding dyes, dyes dependent upon intracellular oxidation,and membrane potential sensitive dyes.

It is understood that the viability stains and viability stainingsystems that selectively label viable cells can use a variety ofprinciples. For example, the fluorescent dyes may penetrate both viableand non-viable cells, but the fluorescent dyes may become modified in aviable cell such that fluorescent dye becomes activated within theviable cell (for example, the activated dye may bind to a cellularsubstrate or cellular organelle or the like, or may become capable offluorescence emission upon illumination with excitation light), or madeinsoluble within the viable cell. The modification can occur as resultof metabolic activity within the cell, for example, via enzymaticactivity within the cell. By way of example, the fluorescent dyeoptionally contains a substrate for an esterase enzyme. Other systemsmay use a combination of a fluorescent dye and a quencher or two or moredifferent quenchers that quench the emission from the fluorescent dyeupon excitation. Other systems may use a plurality of fluorescent dyesor a combination of a dye and quencher, where the emission profile (forexample, emission wavelength) is modulated by the presence of a secondfluorescent dye or the quencher. It is understood that in, each of theforegoing systems, a plurality of different fluorescent dyes can beemployed, for example, 2, 3, 4, or 5 different fluorescent dyes thattarget and/or stain different cells or cell types, organelles,structures, proteins, glycoproteins, lipids, glycolipids, nucleic acids,carbohydrates, etc., to increase confidence that a fluorescent event isactually caused by a cell rather than a non-specific acellular event.

By way of example, viability stains that can be activated within viablecells, for example, via metabolic activity (for example, esteraseenzymatic activity) within the cells, are described in U.S. Pat. Nos.5,314,805, and 5,534,416, U.S. Patent Application Publication No.US2008/0305514, and PCT Publication No. WO92/02632. In one embodiment,the fluorescent dye can contain an esterase substrate. An esterasesubstrate membrane permeable dye becomes chemically modified via anesterase enzyme in a viable cell that creates carboxyl groups that trapthe modified fluorescent dye within intact, viable cells. In thesecases, the background can be reduced by using a membrane impermeablequencher or with a mild detergent to remove any fluorescence fromextracellular regions or non-viable cells. In such an approach, only theviable cells are detected. Exemplary esterase substrate-based membranepermeable dyes are discussed in U.S. Pat. No. 5,534,416, includingwithout limitation, Calcein Blue AM, Carboxycalcein Blue AM, Fluoresceindiacetate, carboxyfluorescein diacetate, 5-carboxyfluoresein diacetateAM, sulfofluorescein diacetate, BCECF-AM, and Calcein AM.

Fluorescent dyes that preferentially label non-viable cells rather thanviable cells (for example, permeate the membranes of non-viable ratherthan viable cells) include for example, PO-PRO-1, BO-PRO-1, YO-PRO-1,TO-PRO-1, PO-PRO-3, BO-PRO-3, YO-PRO-3, TO-PRO-3, POPO-1, BOBO-1,YOYO-1, POPO-3, BOBO-3, YOYO-3 and ethidium bromide (U.S. Pat. No.5,534,416).

In addition, it is completed that the fluorescent dyes useful in thepractice of the invention include dyes that bind to nucleic acids withinthe cells. The dyes can permeate and bind to nucleic acids in viablecells, permeate and bind nucleic acids in non-viable cells, or permeateand bind nucleic acids in viable and non-viable cells. The choice of thedye will depend upon the detection protocol to be employed.

A variety of viability staining systems have been developed and aredescribed, for example, in U.S. Patent Publication 2008/0305514, U.S.Pat. Nos. 5,314,805, and 5,534,416, Canadian Patent application CA02236687, and PCT Publication WO2011/124927, where the viable cells arelabeled with one fluorescent dye, and the non-viable cells are labeledwith a second, different fluorescent dye. In other words, the non-viablecells are still fluorescent, however, the fluorescence emissions fromthe dead cells can be distinguished from the fluorescent emissions fromthe viable cells.

Canadian Patent Application No. 02236687 describes a system that employstwo different fluorescent dyes, a first dye that is capable ofnon-specific transfer across a selective membrane which labels allcells, and a second dye that is incapable of moving across a selectivemembrane and can only enter cells that have compromised membranes. Inthis case, the viable cells are labeled with the first dye and thenon-viable cells are labeled with the first and second dyes. Given thedifferences in the emission spectra of viable and non-viable cells, itis possible to distinguish the viable cells from the non-viable cells.The non-viable cells, however, still generate a fluorescent signal whenirradiated with light of the appropriate wavelength.

U.S. Patent Publication 2008/0305514 describes a system where a firstfluorescent dye, for example, a fluorescent dye containing an esterasesubstrate, permeates and selectively labels viable cells, and a second,different nucleic acid binding fluorescent dye permeates both viable andnon-viable cells and stains the nucleic acid present in both viable andnon-viable cells. In this approach, a fluorescent dye can be, forexample, an esterase substrate that has a high intracellular retentionbecause the esterase in viable cells converts the dye into a form thatcan no longer traverse the cellular membrane. The approach can alsoemploy a nucleic acid stain (for example, a4′,6-diamidino-2-phenylindole dye) that labels both viable andnon-viable cells. When excited with the appropriate wavelengths oflight, the viable cells emit light from both the first and secondfluorescent dyes (i.e., there are two different fluorescent events)whereas the non-viable cells emit light from just the second fluorescentdye. The use of both the viability stain (for example, the esterasesubstrate) and the nucleic acid stain can increase confidence that afluorescent event is actually caused by a cell and not via anon-specific event. The non-viable cells still generated a fluorescentsignal when irradiated with light of the appropriate wavelength.

U.S. Pat. No. 5,314,805 describes a different system that employs twodifferent fluorescent dyes. A first fluorescent dye, for example,calcein AM (an esterase substrate), permeates and selectively labelsviable cells. Viable cells are detectable by a green fluorescent signalgenerated upon enzymatic hydrolysis of calcein AM. Non-viable cells aredetected with a second different fluorescent dye, for example, ethidiumhomodimer. Non-viable cells are detectable by red fluorescence resultingfrom nucleic acids stained with the ethidium homdimer. As a result, itis possible to distinguish between viable and non-viable cells basedupon the emission profile of each cell. The non-viable cells stillgenerate a fluorescent signal when irradiated with light of theappropriate wavelength.

U.S. Pat. No. 5,534,416 describes a different system that employs twodifferent fluorescent dyes. A first fluorescent dye, acyclic-substituted unsymmetrical cyanine dye, stains all cells (bothviable cells and non-viable cells). The second fluorescent dye, is a dyethat selectively labels either viable cells or non-viable cells andgives a fluorescence response that is different from the firstfluorescent dye. When the second fluorescent dye is a dye thatselectively stains viable cells, the viable cells are stained with thefirst and second dyes, and the non-viable cells are stained with thefirst dye. In contrast, when the second fluorescent dye is a dye thatselectively stains non-viable cells, the viable cells are stained withthe first dye and the non-viable cells are labeled with the first andsecond dyes. During either approach, given the differences in theemission spectra of viable and non-viable cells, it is possible todistinguish the viable cells from the non-viable cells. The non-viablecells, however, still generate a fluorescent signal when irradiated withlight of the appropriate wavelength.

In certain staining systems, the fluorescent dyes traverse the intactmembranes of viable cells but become insoluble or trapped within viablecells as a result of metabolic activity within the cells. Furthermore,the fluorescent dyes can be used with one or more quenchers thatselectively enter non-viable cells, which therefore increase theselectivity for a fluorescent event emanating from viable cells. Inother circumstances, the fluorescent dyes may permeate both viable andnon-viable cells. In this scenario, the fluorescent dye can be used withone or more quenchers that selectively permeate non-viable cells, whichtherefore increase selectively for a fluorescent event emanating fromviable cells.

In an alternative approach, U.S. Patent Application Publication No.US2006/0040400 and European Application No. EP 1624 071 describes theuse of a fluorescent dye and a quencher. A fluorescent dye (for example,carboxyfluorescein diacetate) permeates both viable and non-viablecells. The quencher then is added to the fluorescently stained cellsthat is capable of permeating the membrane of a viable cell but does notquench the fluorescence of the fluorescent dye at the pH in the viablecells, but quenches the fluorescence at a pH substantially differentfrom the pH of the viable cells. In this approach, the quencher is addedto the cells at a pH that is substantially different from the pH valueof the viable cells. In this approach, the quencher is not operative toquench fluorescence at the pH of viable cells but quenches fluorescenceat the pH of non-viable cells.

Despite the cell viability stains and viability staining systemsavailable and useful in the practice of the methods and systemsdescribed herein, there is a desire to develop a simple, rapid, robuststaining protocol that can be used to selectively detect viable cells(both single cells and clusters of cells) with little or no backgroundfluorescence emanating from non-viable cells, especially under theconditions (for example, using the excitation light) used to excite theviable cells and/or the system (e.g., membrane, solid support, opticalcell or flow cell) containing or supporting the cells. In addition, thestaining system should not compromise the viability of the cells beingdetected. Furthermore, as discussed below, if desired, the stainingsystem should permit the simultaneous proliferation and staining ofviable cells in the cell sample.

The invention provides a method for selectively labeling viable cells(for example, prokaryotic cells or eukaryotic cells) in a cellcontaining sample. The staining method can be used in combination with acell capture system and/or an optical detection system for detecting thepresence of viable cells in a cell sample. The staining procedure can beused to stain viable cells disposed upon a solid support, for example,microscope slide and/or a well in a culture plate, or within a liquidsample, for example, within an optical cell or a flow cell. The stainingprotocol can be used in a method to measure the bioburden (for example,to measure the number and/or percentage and/or fraction of viable cells(for example, viable microorganisms, for example, bacteria, yeast, andfungi)) of a particular sample of interest.

The invention provides a method of detecting viable cells in a cellsample. The method comprises exposing cells in the cell sample to (i) amembrane permeable fluorescent dye under conditions that permit thefluorescent dye to permeate both viable and non-viable cells, and (ii) amembrane impermeable fluorescence quencher capable of quenchingfluorescence produced by the fluorescent dye under conditions to permitthe quencher to selectively permeate non-viable cells but not viablecells. Thereafter, the method comprises exposing the cells to a beam oflight having a wavelength capable of exciting the fluorescent dye toproduce a fluorescent emission, and detecting the fluorescent emission,if any, from the cells with a detector (e.g., a single detector thatdetects a single wavelength range or a plurality of different wavelengthranges) or a plurality of detectors (e.g., different detectors eachcapable of detecting a different wavelength range), thereby to detectthe viable cells in the cell sample. The fluorescent dye within thenon-viable cells emits substantially less fluorescence than thefluorescent dye within the viable cells. As a result, the non-viablecells emit substantially less fluorescence than the viable cells.

Under certain circumstances, for example, when the quencher is capableof creating fluorescent emissions, the wavelength of the excitationlight can be selected so as not to photoexcite, or substantially not tophotoexcite, the quencher. Alternatively, the detector can be chosenand/or configured so as to preferentially detect the emission eventsfrom the dye rather than the emission events from the quencher.

In addition, the invention provides a method of detecting viable cellsin a cell sample. The method comprises exposing cells in the cell sampleto (i) a membrane permeable fluorescent dye under conditions that permitthe fluorescent dye to permeate both viable and non-viable cells, and(ii) a membrane impermeable fluorescence quencher capable of quenchingfluorescence produced by the fluorescent dye under conditions to permitthe quencher to selectively permeate non-viable cells but not viablecells. Thereafter, the cells are exposed to light having a wavelengthcapable of exciting the fluorescent dye to produce a fluorescentemission. The fluorescent emission, if any, from the cells, is detectedwith one or more detectors configured to preferentially detect adetectable emission from the fluorescent dye rather than a detectableemission from the quencher so that the detectable emission from thequencher, if created, is no greater than 50% (e.g., no greater than 40%,no greater than 30%, no greater than 20%, no greater than 10%) of thedetectable emission from the fluorescent dye. As a result, under thedetection conditions employed, the non-viable cells emit substantiallyless fluorescence detected by the detector than the viable cells. As aresult, the method can be used to detect the viable cells in the cellsample.

In certain embodiments, the non-viable cells emit no or substantially nofluorescence detectable by the detector upon exposure to the light. Incertain other embodiments, the non-viable cells emit no or substantiallyno fluorescence upon exposure to the beam of light.

It is understood that the choice of the appropriate dye-quencher pairdepends upon a number of factors including one or more of: the emissionspectrum of the dye, the absorbance spectrum of the quencher, theemission spectrum of the quencher, if the quencher has an emissionspectrum, and the bandwidth of detection in a given detector. Each ofthese features are discussed below.

In general, exemplary membrane permeable fluorescent dyes useful in thepractice of this method have one or more of the following features:fluorescent when exposed to a beam of light having a wavelength maximain the range of from about 350 nm to about 1000 nm, water soluble withor without a surfactant such as a mild detergent or cyclodextrin carrieragent, non-toxic at concentrations required for detectable staining, anda hydrophobic character that allows for passive diffusion through aviable cell membrane. In certain embodiments, the membrane permeablefluorescent dye may also be characterized as containing at least onecharged group (e.g., a quaternary nitrogen group). In certainembodiments, the fluorescent dye can be excited to undergo fluorescenceby radiation from a red laser, for example, a red laser that emits lighthaving a wavelength in the range of 620 nm to 640 nm.

In addition, exemplary membrane impermeable fluorescent quenchers usefulin the practice of this method have one or more of the followingfeatures: non-toxic at concentrations sufficient for quenching signalfrom the fluorescent dye, water soluble, hydrophilic and polarcontaining either highly charged groups such as but not limited tocarboxyl, amino, phenoxide, sulfate, sulfonate, phosphate, orphosphonate moieties, and/or substituted with polar ligands such aspolyethylene glycol, polypropylene glycol, or polyvinyl alcohol, so thatthe polarity of the quencher prevents passive diffusion through a viablecell membrane and/or is actively pumped out of a viable cell. In certainembodiments, the membrane impermeable fluorescent quencher optionallycontains at least two charged groups (e.g., at least one quaternarynitrogen group). In another embodiment, the quencher does notfluorescence when exposed to light having a wavelength, a wavelengthrange, or a plurality of wavelength ranges, that is emitted from themembrane permeable fluorescent dye when excited by the light source usedfor detecting the presence of viable cells.

Exemplary fluorescent dye and fluorescence quencher pairs can beselected for use in the practice of the inventions described herein andcan be chosen, for example, from the fluorescent dyes and fluorescencequenchers set forth in TABLES I and II below.

Exemplary fluorescent dyes (e⁻ acceptors) can be selected from Oxazine1, Oxazine 170, Oxazine 750, Oxazine 4, Rhodamine 700, Rhodamine 800,Cresyl Violet, Nile blue, Methylene Blue. Azure A, Azure B and Azure C,which can be used in combination with an exemplary quencher (e⁻ donor)selected from sodium ascorbate, 5′guanosine monophosphate, L-tryptophan,potassium hexacyanoferrate (II), diphenylamine-2-sulfonic acid, copperphthalocyanine-3,4′,4″,4′″-tetrasulfonic acid tetrasodium salt and humicacid.

Exemplary fluorescent dyes (e⁻ donors) can be selected from metallatedphthalocyanines or porhyrins, Hoeschst 33342, and other Hoeschst dyes,Ru(phen_dppz²⁺ and Rh(phi)bpy³⁺, which can be used in combination with aquencher (e⁻ acceptor) selected from bipyridinium derivatives (forexample, N,N′-dimethyl-4,4′-bipyridinium dichloride,1,1′-ethylene-2,2′-bipyridyyldiylium dibromide, and anthraquinones (forexample, bisalkylaminoanthraquinones).

TABLE I EXEMPLARY FLUORESCENT DYES Fluorescent Profile ExcitationExemplary (λ max) Excitation Typical Dye Fluorescent Emission WavelengthPET No. Dye Structure Source (λ max) (nm) Quencher D1 Oxazine 1Perchlorate

Sigma- Aldrich, St. Louis, MO Excitation: 643 nm Emission: 665 nm 640 nmElectron donor D2 Oxazine 170 Perchlorate

Sigma- Aldrich, St. Louis, MO Excitation: 613 nm Emission: 641 nm 532nm, 640 nm Electron donor D3 Oxazine 750 Perchlorate

Sigma- Aldrich, St. Louis, MO Excitation: 685 nm Emission: 695 nm 640 nmElectron donor D4 Rhodamine 6G

Sigma- Aldrich, St. Louis, MO Excitation: 524 nm Emission: 552 nm 488nm, 532 nm Electron donor D5 Rhodamine B

Sigma- Aldrich, St. Louis, MO Excitation: 545 nm Emission: 565 nm 488nm, 532 nm Electron donor D6 Rhodamine 700

AnaSpec, Fremont, CA Excitation: 643 nm Emission: 664 nm 640 nm Electrondonor D7 Rhodamine 800

Sigma- Aldrich, St. Louis, MO Excitation: 682 nm Emission: 712 nm 640 nmElectron donor D8 Acridine Orange

Sigma- Aldrich, St. Louis, MO Excitation: 502 nm Emission: 525 nm 488 nmElectron acceptor D9 4′,6- diamidino- 2- phenyl- indole (DAPI)

Sigma- Aldrich, St. Louis, MO Excitation: 358 nm Emission: 461 nm 355 nmElectron acceptor D10 Hoechst 33258

Sigma- Aldrich, St. Louis, MO Excitation: 355 nm Emission: 465 nm 355 nmElectron acceptor D11 Hoechst 33342

Sigma- Aldrich, St. Louis, MO Excitation: 355 nm Emission: 465 nm 355 nmElectron acceptor D12 BOXTO TATAA Excitation: 488 nm Electron Biocenter,San 515 nm acceptor Francisco, CA Emission: 552 nm D13 Vybrant LifeExcitation: 365 nm, Electron DyeCycle Technologies 369 nm 405 nmacceptor Violet Inc., Grand Emission: Island, NY 437 nm D14 Vybrant LifeExcitation: 488 nm Electron DyeCycle Technologies 506 nm acceptor GreenInc., Grand Emission: Island, NY 534 nm D15 Vybrant Life Excitation: 488nm, Electron DyeCycle Technologies 519 nm 532 nm acceptor Orange Inc.,Grand Emission: Island, NY 563 nm D16 Vybrant Life Excitation: 488 nm,Electron DyeCycle Technologies 638 nm 532 nm, acceptor Ruby Inc., GrandEmission: 633 nm Island, NY 686 nm D17 Draq5

BioStatus Limited, Leicestershire, UK Excitation: 488-647 nm Emission:697 nm 488 nm, 532 nm, 633 nm Electron donor D18 CYTRAK BioStatusExcitation: 488 nm, Electron Orange Limited, 515 nm 532 nm donorLeicestershire, Emission: UK 605 nm D19 Tris (bipyridine) ruthenium (II)dichloride

Sigma- Aldrich, St. Louis, MO Excitation: 452 nm Emission: 623 nm 355nm, 488 nm Electron acceptor D20 Cresyl Violet Perchlorate

Sigma- Aldrich, St. Louis, MO Excitation: 602 nm Emission: 614 nm 532 nmElectron donor D21 SYTO 9 Life Excitation: 488 nm Electron Technologies485 nm acceptor Inc., Grand Emission: or donor Island, NY 498 nm D22SYTO 11 Life Excitation: 488 nm Electron Technologies 508 nm acceptorInc., Grand Emission: or donor Island, NY 527 nm D23 SYTO 12 LifeExcitation: 488 nm Electron Technologies 499 nm acceptor Inc., GrandEmission: or donor Island, NY 522 nm D24 SYTO 13 Life Excitation: 488 nmElectron Technologies 488 nm acceptor Inc., Grand Emission: or donorIsland, NY 509 nm D25 SYTO 14 Life Excitation: 488 nm ElectronTechnologies 517 nm acceptor Inc., Grand Emission: or donor Island, NY549 nm D26 SYTO 16 Life Excitation: 488 nm Electron Technologies 488 nmacceptor Inc., Grand Emission: or donor Island, NY 518 nm D27 SYTO 18Life Excitation: 488 nm Electron Technologies 490 nm acceptor Inc.,Grand Emission: or donor Island, NY 507 nm D28 SYTO 21 Life Excitation:488 nm Electron Technologies 494 nm acceptor Inc., Grand Emission: ordonor Island, NY 517 nm D29 SYTO 24 Life Excitation: 488 nm ElectronTechnologies 490 nm acceptor Inc., Grand Emission: or donor Island, NY515 nm D30 SYTO 25 Life Excitation: 488 nm, Electron Technologies 521 nm532 nm acceptor Inc., Grand Emission: or donor Island, NY 556 nm D31SYTO BC Life Excitation: 488 nm Electron Technologies 485 nm acceptorInc., Grand Emission: or donor Island, NY 500 nm D32 SYTO 80 LifeExcitation: 488 nm, Electron Technologies 531 nm 532 nm acceptor Inc.,Grand Emission: or donor Island, NY 545 nm D33 SYTO 81 Life Excitation:488 nm, Electron Technologies 530 nm 532 nm acceptor Inc., GrandEmission: or donor Island, NY 544 nm D34 SYTO 82 Life Excitation: 488nm, Electron Technologies 541 nm 532 nm acceptor Inc., Grand Emission:or donor Island, NY 560 nm D35 SYTO 83 Life Excitation: 488 nm, ElectronTechnologies 543 nm 532 nm acceptor Inc., Grand Emission: or donorIsland, NY 559 nm D36 SYTO 84 Life Excitation: 488 nm, ElectronTechnologies 567 nm 532 nm acceptor Inc., Grand Emission: or donorIsland, NY 582 nm D37 SYTO 85 Life Excitation: 488 nm, ElectronTechnologies 567 nm 532 nm acceptor Inc., Grand Emission: or donorIsland, NY 583 nm D38 SYTO 17 Life Excitation: 580 nm ElectronTechnologies 621 nm acceptor Inc., Grand Emission: or donor Island, NY634 nm D39 SYTO 59 Life Excitation: 580 nm, Electron Technologies 622 nm633 nm acceptor Inc., Grand Emission: or donor Island, NY 645 nm D40SYTO 60 Life Excitation: 633 nm Electron Technologies 652 nm acceptorInc., Grand Emission: or donor Island, NY 678 nm D41 SYTO 61 LifeExcitation: 580 nm, Electron Technologies 628 nm 633 nm acceptor Inc.,Grand Emission: or donor Island, NY 645 nm D42 SYTO 62 Life Excitation:633 nm Electron Technologies 652 nm acceptor Inc., Grand Emission: ordonor Island, NY 676 nm D43 SYTO 63 Life Excitation: 633 nm ElectronTechnologies 657 nm acceptor Inc., Grand Emission: or donor Island, NY673 nm D44 SYTO 64 Life Excitation: 580 nm Electron Technologies 599 nmacceptor Inc., Grand Emission: or donor Island, NY 619 nm

TABLE II EXEMPLARY FLUORESCENCE QUENCHERS FRET Quenching Quencher PETApplicable No. Quencher Structure Source Profile Range Q1 SodiumAscorbate

Sigma- Aldrich, St. Louis, MO Electron donor N/A Q2 Potassium hexacyano-ferrate (II)

Sigma- Aldrich, St. Louis, MO Electron donor N/A Q3 Guanosine 5′- mono-phosphate sodium salt

Sigma- Aldrich, St. Louis, MO Electron donor N/A Q4 Sodium diphenyl-amine-4- sulfonate

Sigma- Aldrich, St. Louis, MO Electron donor N/A Q5 4- Sulfocalix[6]arene

Tokyo Chemical Industry Co., Ltd., Portland, OR Electron donor N/A Q6 4-Sulfocalix [8]arene

Tokyo Chemical Industry Co., Ltd., Portland, OR Electron donor N/A Q7Methyl viologen

Sigma- Aldrich, St. Louis, MO Electron acceptor N/A Q8 Sodium 2- anthra-quinone- sulfonate

Sigma- Aldrich, St. Louis, MO Electron acceptor N/A Q9 Potassiumhexacyano- ferrate (III)

Sigma- Aldrich, St. Louis, MO Electron acceptor N/A Q10 Tartrazine

Sigma- Aldrich, St. Louis, MO N/A 400-500 nm Q11 Amaranth

Sigma- Aldrich, St. Louis, MO N/A 500-600 nm Q12 Trypan Blue

Sigma- Aldrich, St. Louis, MO N/A 550-650 nm Q13 Naphthol Green B

Sigma- Aldrich, St. Louis, MO N/A 600-750 nm Q14 IR-783

Sigma- Aldrich, St. Louis, MO N/A 600-780 nm Q15 Copper phthalo-cyanine- 3,4′,4″,4′′′- tetra- sulfonic acid tetra- sodium salt

Sigma- Aldrich, St. Louis, MO Electron donor and acceptor 500-700 nm Q16(3-{[4- chloro- 9,10- dioxo-5,8- bis({[3- (trimethyl- ammonio) propyl]amino}) anthracen- 1- yl]amino} propyl) trimethyl- azanium- trihalide

  X⁻ = Cl⁻, Br⁻, or I⁻ Electron acceptor 400-750 nm Q17 (3-{[9,10-dioxo- 4,5,8- tris({[3- (trimethyl- ammonio) propyl] amino}) anthracen-1- yl]amino} propyl) trimethyl- azanium- tetrahalide

  X⁻ = Cl⁻, Br⁻, or I⁻ Electron acceptor 500-800 nm Q18 N-methyl- 4-[(4-{methyl[3- (trimethyl- ammonio) propyl] amino} phenyl)

  X⁻ = Cl⁻, Br⁻, or I⁻ Electron acceptor 550-800 nm imino]- N-[2-(trimethyl- ammonio) ethyl] cyclohexa- 2,5-dien-1- Iminium trihalide Q19Draq7 BioStatus Electron 400-640 nm Limited, acceptor Leicestershire, UK

The membrane impermeable quenchers for use in the practice of theinventions described herein typically suppress fluorescence from thefluorescent dye by either photo-induced electron transfer (PET),sometimes referred to as static quenching, or fluorescence resonanceenergy transfer (FRET), or some combination thereof. The efficiency ofthese quenching mechanisms are distance-dependent, meaning that thefluorescent dye molecule and quencher molecule must be in close enoughproximity for the suppression of fluorescence to take place. It ispossible to select the conditions to maximize the chances that efficientquenching can be accomplished. An exemplary, spectral overlap betweenthe fluorescent dye and quencher is shown schematically in FIG. 12A. Insuch a system, the fluorescent emission of the fluorescent dye issuppressed by the quencher via a FRET mechanism. Using such principlesit is possible to create fluorescent dye-quencher pairs useful in thepractice of the invention.

In one approach, the fluorescent dye-quencher pairs can be selected tohave a binding affinity for one another, by means of, for example,electrostatic and/or hydrophobic interactions, which when bound resultsin a substantially non-fluorescent ground-state complex. In other words,the fluorescent dye and fluorescence quencher may bind to one another inthe cell.

Exemplary combinations of fluorescent dyes and fluorescence quenchersuseful in such an approach are set forth in TABLE III.

TABLE III Exemplary Dye—Quencher Combinations Exemplary Dye ExemplaryQuencher D1 Q1, Q2, Q3, Q4, Q5, Q6, Q8, Q13, Q14, or Q15, or anycombination thereof D2 Q1, Q2, Q3, Q4, Q5, Q6, Q8, Q13, Q14, or Q15, orany combination thereof D3 Q1, Q2, Q3, Q4, Q5, Q6, Q8, Q13, Q14, or Q15,or any combination thereof D4 Q1, Q2, Q3, Q4, Q5, Q6, Q8, Q11, Q12, orQ15, or any combination thereof D6 Q1, Q2, Q3, Q4, Q5, Q6, Q8, Q13, Q14,or Q15, or any combination thereof D7 Q1, Q2, Q3, Q4, Q5, Q6, Q8, Q13,Q14, or Q15, or any combination thereof D8 Q2, Q5, Q6, Q11, or Q12, orany combination thereof D20 Q1, Q2, Q3, Q4, Q5, Q6, Q8, Q13, Q14, orQ15, or any combination thereof

In another approach, the fluorescent dye and/or quencher can be selectedto have binding affinities for a specific cellular component, organelle,or structure, such as a nucleic acid. For example, the fluorescent dye,the fluorescence quencher, or both bind to a nucleic acid in a cell.When the fluorescent dye and quencher are co-bound to the specifictarget, such as a nucleic acid, the proximity of the dye and quencherare such that a substantially non-fluorescent complex is formed. Ineither approach, the membrane impermeable quencher is not permitted toenter viable cells via an intact membrane, which prevents the formationof the non-fluorescent complex in viable cells. In one embodiment of theinvention, fluorescent dye—fluorescence quencher pairs are chosen suchthat the fluorescent dye and fluorescence quencher both bind to aspecific cellular component such as a nucleic acid. This approach hasthe advantage that non-specific fluorescent staining is reduced becausethe fluorescent dye and fluorescence quencher bind to a specific target.As a result, this approach can reduce non-specific fluorescent stainingwhich optimizes the signal to noise ratio when differentiating betweenviable cells. The nucleic acid binding fluorescent dyes and nucleic acidbinding fluorescence quenchers are selected such that there is spectraloverlap between the emission spectrum of the fluorescent dye and theabsorbance spectrum of the quencher, for example, as shown schematicallyin FIG. 12A.

Exemplary combinations of fluorescent dyes and fluorescence quenchersthat selectively bind a nucleic acid (for example, a DNA or RNA) are setforth in TABLE IV. The feature of spectral overlap between the emissionspectrum of the fluorescent dye and the absorbance spectrum of thequencher may be used to guide the selection of a particular fluorescentdye for use with a particular quencher. This approach can beparticularly effective when the dye and quencher both bind, for example,selectively bind to a cellular organelle or cell component such as aprotein, glycoprotein, carbohydrate, lipid, or nucleic acid.

TABLE IV Exemplary Nucleic Acid Binding Dye—Nucleic Acid BindingQuencher Combinations Exemplary Nucleic Acid Binding Dye ExemplaryNucleic Acid Binding Quencher D1 Q16, Q17, or Q18, or any combinationthereof D2 Q16, Q17, or Q18, or any combination thereof D3 Q16, Q17, orQ18, or any combination thereof D4 Q16, Q17, or Q19, or any combinationthereof D6 Q16, Q17, or Q18, or any combination thereof D7 Q16, Q17, orQ18, or any combination thereof D8 Q16, Q17, or Q19, or any combinationthereof D9 Q16, Q17, or Q19, or any combination thereof D10 Q16, Q17, orQ19, or any combination thereof D11 Q16, Q17, or Q19, or any combinationthereof D12 Q16, Q17, or Q19, or any combination thereof D13 Q16, Q17,or Q19, or any combination thereof D14 Q16, Q17, or Q19, or anycombination thereof D15 Q16, Q17, or Q19, or any combination thereof D16Q16, Q17, or Q18, or any combination thereof D17 Q16, Q17, or Q18, orany combination thereof D18 Q16, Q17, or Q19, or any combination thereofD19 Q16, Q17, Q18, or Q19, or any combination thereof D70 Q16, Q17, Q18,or Q19, or any combination thereof D21 Q16, Q17, or Q19, or anycombination thereof D22 Q16, Q17, or Q19, or any combination thereof D23Q16, Q17, or Q19, or any combination thereof D24 Q16, Q17, or Q19, orany combination thereof D25 Q16, Q17, or Q19, or any combination thereofD26 Q16, Q17, or Q19, or any combination thereof D27 Q16, Q17, or Q19,or any combination thereof D28 Q16, Q17, or Q19, or any combinationthereof D29 Q16, Q17, or Q19, or any combination thereof D30 Q16, Q17,or Q19, or any combination thereof D31 Q16, Q17, or Q19, or anycombination thereof D32 Q16, Q17, or Q19, or any combination thereof D33Q16, Q17, or Q19, or any combination thereof D34 Q16, Q17, or Q19, orany combination thereof D35 Q16, Q17, or Q19, or any combination thereofD36 Q16, Q17, or Q19, or any combination thereof D37 Q16, Q17, or Q19,or any combination thereof D38 Q16, Q17, Q18, or Q19, or any combinationthereof D39 Q16, Q17, Q18, or Q19, or any combination thereof D40 Q16,Q17, or Q18, or any combination thereof D41 Q16, Q17, Q18, or Q19, orany combination thereof D42 Q16, Q17, or Q18, or any combination thereofD43 Q16, Q17, or Q18, or any combination thereof D44 Q16, Q17, Q18, orQ19, or any combination thereof

The dye and quencher can be chosen to permit spectral overlap of theemission spectrum of the dye with the absorption spectrum of thequencher, for example, as demonstrated in FIG. 12A to permit therequisite level of quenching to occur. Under these conditions, thefluorescent emission of the fluorescent dye can be suppressed by thequencher via FRET.

The dye and quencher pair can be further characterized according to theextent to which the emission spectrum of the dye overlaps the emissionspectrum (if any) of the quencher. Under certain circumstances, it canbe desirable to minimize the extent to which emission spectrum of thequencher overlaps with the emission spectrum of the fluorescent dye,particularly that portion of the emission spectrum of the dye that isdetected by a detector (e.g., a fluorescence detector) during the cellviability assay. Therefore, in certain embodiments, the quencher isselected such that if the quencher is excited by the light source(excitation light) used to excite the fluorescent dye and such quencheris capable of emitting a photon, then the quencher preferably has one ormore of the following properties: (i) the quencher does not emitsubstantial, or any, electromagnetic radiation within the emissionspectrum of the dye (particularly that portion of the emission spectrumof the fluorescent dye that is measured during the cell viability assay,i.e., the portion of the emission spectrum of the fluorescent dye thatfalls within the bandwidth of detection in the detector (see, wavelengthrange λ₁-λ₂ in FIG. 12B)), and (ii) the quencher has a lower quantumyield than that of the fluorescent dye. As a result, the quencherremains non-fluorescent or substantially non-fluorescent over the rangeof wavelengths measured during the cell viability assay. Accordingly,when a detection channel in a detector is set to detect the emissionsignal emanating preferentially from the fluorescent dye, substantiallyless fluorescence (for example, less than 40%, 30%, 20%, 10% or 5% ofthe fluorescence) or no fluorescence emanating from the quencher (forexample, via auto-fluorescence or fluorescence created by the excitationsource) is detected in the detection channel of the detector (see,wavelength range λ₁-λ₂ in FIG. 12B).

In one approach for selecting an appropriate dye-quencher pair, as shownschematically in FIG. 12B, the emission spectrum of the fluorescent dye(e.g., the membrane permeable dye) is shown by the hatched line and theemission spectrum of the quencher (e.g., the membrane impermeablequencher) is shown by the solid line. A detector with a bandwidth ofdetection (BD, which is represented, for example, as a wavelength rangebetween wavelength value λ₁ and wavelength value λ₂) is capable ofdetecting a portion of the emission spectrum of the fluorescent dye(area denoted as region X). The area X is the detectable emission of thefluorescent dye. Given the bandwidth of detection, the detector issometimes also capable of detecting a portion of the emission spectrumof the quencher (area denoted as region Y), assuming that the quencherhas its own emission spectrum (certain quenchers are not fluorescent anddo not produce an emission spectrum) or the emission spectrum of thequencher overlaps at least a portion of the emission spectrum of thedye. The area Y is the detectable emission of the quencher. In caseswhere the quencher does produce an emission spectrum, the detectableemission of the quencher (area Y) is no greater than 50% (e.g., nogreater than 40%, no greater than 30%, no greater than 20%, no greaterthan 10%, or no greater than 5%) of the detectable emission of the dye(area X) so as to provide the appropriate signal-to-noise ratio for thedetection of viable cells. In a preferred embodiment, the detectableemission of the quencher (area Y) is no greater than 30% of thedetectable emission of the dye (area X).

This approach can be performed by taking the fluorescence emissionspectra of the dye and quencher and then identifying the portions (areasunder the curve) under each spectra that fall within the bandwidth ofthe proposed detector (see FIG. 12B). The ratio of these values is thenused to determine that the detectable emission of the quencher is nogreater than 50% of the detectable emission of the dye. The choice ofthe dye-quencher pair depends upon the choice of the appropriatebandwidth of detection in the detector, the fluorescent dye and thequencher.

An alternative approach can be used for selecting an appropriatedye—quencher pair. For example, in another embodiment, the separation ofthe emission spectra of the fluorescent dye from the emission spectra(if present) of the quencher can be characterized according tonon-overlap of the critical emission wavelength range of the fluorescentdye and the critical emission wavelength range of the quencher. Theseparation of the critical emission wavelength range of the fluorescentdye from the critical emission wavelength range of the quencher isillustrated in FIG. 12C. The term “critical emission wavelengthrange(s)” or CER_(λ) means the wavelength range(s) of the emissionprofile over which a fluorophore has an emission intensity that is atleast 40%, 50%, 60%, 70%, 80%, or 90% of the maximum emission intensityat the λ_(max) of the emission spectrum of the fluorophore. Thus, thecritical emission wavelength range of the fluorescent dye is thewavelength range of the emission profile of the fluorescent dye overwhich the dye has an emission intensity that is at least 40%, 50%, 60%,70%, 80%, or 90% of the maximum emission intensity at the λ_(max) of theemission spectrum of the fluorescent dye. The critical emissionwavelength range of the quencher is the wavelength range of the emissionprofile (if any) of the quencher over which the quencher has an emissionintensity that is at least 40%, 50%, 60%, 70%, 80%, or 90% of themaximum emission intensity at the λ_(max) of emission spectrum of thequencher.

To illustrate further, the emission intensity of an exemplaryfluorescent dye and emission intensity of an exemplary quencher areillustrated in FIG. 12C. The critical emission wavelength range of thefluorescent dye is labeled CER_(λ) dye, and corresponds to thosewavelengths where the emission intensity of the dye is at least 50% ofthe maximum emission intensity of the dye at its λ_(max). The criticalemission wavelength range of the quencher is labeled CER_(λ) quencher,and corresponds to those wavelengths where the emission intensity of thequencher is at least 50% of the maximum emission intensity of thequencher at its λ_(max). As depicted in FIG. 12C, there is no overlap ofthe critical emission wavelength range of the fluorescent dye with thecritical emission wavelength range of the quencher. The differencebetween the wavelengths (i.e., λ₁-λ₂) is denoted by the term “d” in FIG.12C, which preferably is at least 5 nm (or at least 10 nm, 20 nm, 30 nm,40 nm, 50 nm, 60 nm, 70 nm, 80 nm, 90 nm or 100 nm).

Depending on the emission spectra of the fluorescent dye and thequencher, it is understood that wavelengths that exceed the thresholdintensity (e.g., 50% of the maximum emission intensity of the dye at itsλ_(max)) may not be continuous. For example, the emission spectra of thedye and/or quencher may contain two or more wavelength ranges thatexceed the threshold intensity (e.g., 50% of the maximum emissionintensity of the dye or the quencher at its respective λ_(max)).Preferably there is little or no overlap between the critical emissionwavelength range(s) of the fluorescent dye and the critical emissionwavelength range(s) of the quencher. Using these criteria, it ispossible to choose dye-quencher combinations useful in the cellviability assays described herein.

The concentrations of a particular membrane permeable fluorescent dyeand membrane impermeable fluorescent quencher are selected to (i)provide sufficient fluorescence intensity from the fluorescent dye inviable cells, (ii) substantially quench fluorescence of the fluorescentdye in non-viable cells, and (iii) to minimize any toxicity of the dyeand/or quencher. In certain embodiments, the membrane permeablefluorescent dye is used at a concentration in the range of from about0.1 μM to about 50 μM, from about 0.5 μM to about 30 μM, or from about 1μM to about 10 μM, when applied to cells. In certain embodiments, theamount of membrane impermeable fluorescent quencher applied to cells isabout the same molar concentration as the fluorescent dye or greater. Incertain embodiments, the amount of membrane impermeable fluorescentquencher applied to cells is at a concentration of 0.1 μM to about 200mM, from about 0.5 μM to about 100 mM, from about 0.5 μM to about 1 mM,from about 0.5 μM to about 500 μM, from about 1 μM to about 500 μM, orfrom about 1 μM to about 100 μM, when applied to cells. In otherembodiments, the quencher is provided at a molar excess of, for example,2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold,20-fold, 40-fold, 60-fold, 80-fold, 100-fold, 200-fold, 300-fold,400-fold, 500-fold, 1000-fold, or 2,000-fold, 5,000-fold, 10,000-fold,20,000-fold, 50,000-fold or 100,000-fold, relative to the concentrationof the fluorescent dye. The amount of the quencher should be chosen sothat the quencher is non-toxic or is substantially non-toxic to thecells being detected in a given sample. In certain embodiments, theamount of membrane impermeable fluorescent quencher applied to cells isin the range of about a 5-fold molar excess to about a 20-fold molarexcess or about a 5-fold molar excess to about a 10-fold molar excess,relative to the amount of membrane permeable fluorescent dye applied tocells.

Depending upon the cells being detected and desired sensitivity, it ispossible to expose the cells to a plurality of different fluorescentdyes and/or a plurality of different fluorescence quenchers.Furthermore, depending upon the dyes and quenchers to the used, and thecells to be detected, the cells can be exposed to the fluorescent dyeand then exposed to fluorescence quencher. Alternatively, the cells canbe exposed to the fluorescent dye and the fluorescence quencher at thesame time. In certain methods, a washing step using, for example, aphysiologically acceptable salt and/or buffer solution can be used toremove residual dye or quencher before detection. In certain methods, asubsequent washing step is not necessary and the washing step ifemployed could even be undesirable if the fluorescent dye is removed orextracted from the viable cells, for example, via diffusion out of thecells during the washing step.

The detection method can be performed on single cells, clusters of cellsor colonies of cells. Under certain circumstances, for example, toincrease the sensitivity of the assay, it may be desirable to culturethe cells under conditions that permit cell proliferation prior toand/or during and/or after exposing the cells to the fluorescent dye andthe fluorescence quencher. The culture conditions, including, the choiceof the growth media, the temperature, the duration of the culture, canbe selected to permit at least one of cells in the sample to have one ormore cell divisions.

For example, depending upon the sensitivity required, the cells, oncecaptured on the membrane, can be contacted with growth media and/orspore germination initiators and then permitted to proliferate for oneor more doubling times to increase the number of cells at a particularlocus on the membrane. In one embodiment, the cells are captured onmembrane 202, a solution containing the fluorescent dye and thefluorescence quencher and growth medium (e.g., Nutrient Broth T7 105from PML Microbiologicals, Wisonville, Oreg.) are poured into the cupassembly and pulled through membrane 202 via vacuum suction. The lid 588is then placed upon base 554 (see FIG. 10C), and the resulting unit canbe placed in an incubator at a preselected temperature (e.g., 37° C.)for a desired length of time (e.g., from 15 minutes to 8 hours, or from30 minutes to 4 hours) depending upon the doubling time of theorganisms. During this time, the membrane 202 remains moist in view ofthe growth media and stain present within solid support 558. Thisapproach also provides more time for the fluorescent dye and quencher topermeate and stain the cells. After incubation, the base 554 can then betransferred to and placed into stage 802 for insertion into thedetection device.

In certain embodiments, the fluorescent dye and/or the fluorescencequencher, bind to a nucleic acid within the cell. In other embodiments,the fluorescent dye and the fluorescence quencher bind to one another inthe cell.

In certain embodiments, the beam of light used to excite the fluorescentdye or fluorescent dyes has a wavelength in the range of from about to350 nm to about 1000 nm, from about 350 nm to about 900 nm, from about350 nm to about 800 nm, from about 350 nm to about 700 nm, or from about350 nm to about 600 nm. For example, the wavelength of excitation lightis at least in one range from about 350 nm to about 500 nm, from about350 nm to about 500 nm, from about 350 nm to about 600 nm, from about400 nm to about 550 nm, from about 400 nm to about 600 nm, from about400 nm to about 650 nm, from about 450 nm to about 600 nm, from about450 nm to about 650 nm, from about 450 nm to about 700 nm, from about500 nm to about 650 nm, from about 500 nm to about 700 nm, from about500 nm to about 750 nm, from about 550 nm to about 700 nm, from about550 nm to about 750 nm, from about 550 nm to about 800 nm, from about600 nm to about 750 nm, from about 600 nm to about 800 nm, from about600 nm to about 850 nm, from about 650 nm to about 800 nm, from about650 nm to about 850 nm, from about 650 nm to about 900 nm, from about700 nm to about 850 nm, from about 700 nm to about 900 nm, from about700 nm to about 950 nm, from about 750 to about 900 nm, from about 750to about 950 nm or from about 750 to about 1000 nm. Certain rangesinclude from about 350 nm to about 600 nm and from out 600 nm to about750 nm.

The fluorescent emission can be detected within a range of from about350 nm to about 1000 nm, from about 350 nm to about 900 nm, from about350 nm to about 800 nm, from about 350 nm to about 700 nm, or from about350 nm to about 600 nm. For example, the fluorescent emission can bedetected within a range from about 350 nm to 550 nm, from about 450 nmto about 650 nm, from about 550 nm to about 750 nm, from about 650 nm toabout 850 nm, or from about 750 nm to about 950 nm, from about 350 nm toabout 450 nm, from about 450 nm to about 550 nm, from about 550 nm toabout 650 nm, from about 650 nm to about 750 nm, from about 750 nm toabout 850 nm, from about 850 nm to about 950 nm, from about 350 nm toabout 400 nm, from about 400 nm to about 450 nm, from about 450 nm toabout 500 nm, from about 500 nm to about 550 nm, from about 550 nm toabout 600 nm, from about 600 nm to about 650 nm, from about 650 nm to700 nm, from about 700 nm to about 750 nm, from about 750 nm to about800 nm, from about 800 nm to about 850 nm, from about 850 nm to about900 nm, from about 900 nm to about 950 nm, or from about 950 nm to about1000 nm. In certain embodiments, the emitted light is detected in therange from about 660 nm to about 690 nm, from about 690 nm to about 720nm, and/or from about 720 nm to about 850 nm.

In each of the foregoing, the method can further comprise exposing thecells to a second, different membrane permeable fluorescent dye thatlabels viable cells, non-viable cells or a combination of viable andnon-viable cells.

(IV) Cell Detection

Once the cell capture system has been used to capture cells originallypresent in the fluid sample, the membrane or the membrane assembly canbe inserted into a membrane holder (e.g., holder 802) for insertion intoa suitable detection system. Exemplary detection systems are described,for example, in International Patent Application No. PCT/IB2010/054965,filed Nov. 3, 2010, U.S. patent application Ser. No. 13/034,402, filedFeb. 24, 2011, International Patent Application No. PCT/IB2010/054966,filed Nov. 3, 2010, U.S. patent application Ser. No. 13/034,380, filedFeb. 24, 2011, International Patent Application No. PCT/IB2010/054967,filed Nov. 3, 2010, and U.S. patent application Ser. No. 13/034,515,filed Feb. 24, 2011. In the foregoing detection systems, a membrane isrotated while a beam of excitation light is directed onto the surface ofthe membrane. The emitted light is detected with at least one opticaldetector.

In order to facilitate rotation of the permeable membrane, the membranecan be disposed in a membrane holder. In one embodiment, for example, ina membrane assembly that comprises a mask and optional spokes, themembrane assembly may be inserted into a membrane holder that can beplaced within sample assembly 120 of FIG. 1A. In particular, themembrane assembly maybe placed upon the rotating platform 130.

FIGS. 13A and 13B show an exemplary membrane holder that can be used insuch a detection system. Membrane holder 700 comprises a container 702(e.g., a metallic container made of aluminum) defining a centralcylindrical recess 704 and an offset drive aperture or notch 706. Thecontainer 702 may be disposed upon a rotatable shaft such that the shaftis received within, coupled to, or otherwise engaged with the recess704. The shaft may form a disk to support the holder 700 and can includea protrusion, such as a driver pin that couples with the drive notch706. As a result, rotation of the disk about its axis of rotationcorrespondingly positively rotates the membrane holder 700 withoutslippage.

The container 702 also defines a chamber 708 to receive a membrane andany components for holding the membrane generally flat. These componentsinclude a holder having spokes (described with reference to FIGS. 2A and2B), the masks (described with reference to FIGS. 3A and 3B), and/or orthe porous supporting member (described with reference to FIGS. 4A-4B).Under the chamber 708, a plurality of magnets 712 can be disposed withinthe container 702. An exemplary magnet configuration 710 is depicted inFIG. 13B. The configuration 710 includes three magnets 712 locatedapproximately in a circular pattern having a center at or near the axisof rotation of the container 702. The plane of the circle of magnets 712is substantially parallel to the surface of the chamber 708. Thecontainer 702 further comprises a window 714 such as a glass,polycarbonate or PERSPEX® window enclosed in a magnetic ring 716. Incertain embodiments, the magnets 712 are disk magnets and are used tomaintain the elements in the chamber 708 during rotation (e.g., byattraction of the magnetic ring 716). It should be understood that theconfiguration 710 is for illustrative purposes only, and that otherconfigurations, such as those having fewer or more than three magnets,may incorporate patterns other than a circular pattern, as well as otherretention schemes, and are considered within the scope of the presentinvention.

The window 714 protects the underlying cell retaining membrane, as wellas the cells, and can maintain the sterility of the membrane if is to besubsequently removed and incubated under conditions (e.g., temperature,moisture, and nutrition) to facilitate growth of the viable cells. Themagnetic ring 716 can have an extension 718 forming a magnetic stainlesssteel ring 720. The center of the extension 718 is located at or nearthe axis of rotation of container 702. Accordingly, when the window 714is disposed over a membrane assembly received in the chamber 708, thering 720 is substantially disposed directly over the configuration 710of magnets 712. As the magnetic ring 720, and hence, the window 714 aremoved toward the magnets 712, the membrane received in the chamber 708is generally held in place as the container 702 rotates about its axisof rotation. The extension 718 may also apply downward pressure on themembrane (e.g., via a central mask, etc.), helping preserve the flatnessof the membrane received in the chamber 708.

FIG. 13C depicts a membrane holder assembly for use in an opticaldetection system assembly disposed within the container (also called acartridge holder) 702. Other membrane assemblies, including thosedescribed herein, may also be received in the container/holder 702. Thewindow 714 is disposed over the membrane assembly 200. The magnets 712are disposed in recesses 770 in a bottom surface 772 of the container702. As described above, when the membrane assembly 200 is received inthe chamber 708, the magnets 712 pull ring 720 of the window 714 towardthe surface 772 of the container/cartridge holder 702, thereby holdingthe membrane assembly 200 in place, as depicted in FIG. 13D.

The container/holder 702 can then be placed on a disk or chuck 780 thathas a shaft 782 and a driver mechanism 784 that engages a recess definedby the base of the container/holder 702. The shaft 782 engages with thenotch 704. The disk/chuck 780 fits on a motor shaft of the detectionsystem. Rotation of the motor shaft drives the rotation of the membraneassembly 200. The shaft 782 and the driver 784 prevent thecontainer/holder 702 from slipping or sliding on the surface of the disk780. In addition, the magnets 786 align the container/cartridge holder702 with a predetermined position on the surface of the disk 780,thereby facilitating registration of the initial orientation of themembrane assembly 200. Such registration can be beneficial when mappingthe location of any fluorescence events (e.g., light emitted by viablecells, non-viable cells or particles).

Many other embodiments of a membrane holder are contemplated. Forexample, a membrane holder with three magnets disposed in a rim of acontainer and a window having an integral or separate magnetic rim thathas three notches and three legs has been contemplated. In such anembodiment, the rim of the membrane holder may have three stops. Whenthe magnetic rim is disposed over the container, the legs can slidealong the outer surface of the rim until the legs are in contact withthe stops. The magnets are disposed in the rim and the notches arelocated in the magnetic rim, such that when the legs are in contact withthe stoppers, each of the notches is positioned directly over one of themagnets. In this position, the magnetic rim and the window can bereadily separated from the membrane holder, and a membrane assembly canbe received in or removed from a chamber of the container. Thereafter,the window can be replaced. The window can be rotated such that thenotches are not aligned with the magnets, and as such, the magnetic rimand the window are substantially held in place by the magnets.Consequently, the membrane assembly received in the chamber is alsosubstantially held in place.

In order to minimize the number of manipulation steps for transferringthe porous membrane assembly 200 into the membrane holder, which canincrease the risk of contaminating the membrane assembly 200, it iscontemplated that the membrane holder can be adapted to engage themembrane assembly together with the base of a cup (e.g., the base 554).FIGS. 14A-14C depict the stage 802 adapted to receive the base 554. Thestage 802 may have multiple recesses 810, each adapted to receive aseparate base 554. Walls 812 of the recesses are tapered to receive thesimilar tapered lower portion 576 of the base 554, helping ensure asecure fit for stability during rotation. The stage 802 is depicted in asubstantially circular form, but may be any shape. The stage 802 may besized to fit within the enclosure 110, either permanently ortemporarily. A lower surface of the stage 810 includes a mating recess814 for attachment to the chuck 804 (depicted in FIGS. 15A-15D). Thechuck 804 provides a base on which the stage 802 sits, and provides themeans for rotating the stage 802. The chuck 804 can be permanentlyinstalled in the enclosure 110, or may be removable. In an embodimentwhere the stage 802 and the chuck 804 are already disposed within theenclosure 110, only the base 554 with the saturated membrane 202 wouldneed to be transferred into the enclosure 110 to begin operation, thusminimizing the number of handling steps. The chuck 804 has an uppersurface 820 that can be sized to support a large portion of the stage802 for increased stability during operation. A protrusion 822 on thetop surface 820 is adapted to mate with the mating recess 814 of thestage 802, which is depicted in broken outline in FIG. 15A. Theprotrusion 822 may have an aperture 824 for receiving a fastener (e.g.,a set screw) for further securing the stage 802 to the chuck 804. Abottom surface 826 of the chuck 804 has a protrusion 828 for mating witha drive for rotating the chuck 804.

As discussed above, for accurate detection and/or estimation of cellsand/or particles, the porous membrane 202 should be flat, substantiallyhorizontal, and at or about a predetermined distance from the source ofthe light impinged thereupon. Optionally, the membrane 202 is located ator near the focal length of the detection system 170. The thickness ofthe base 554 and flatness of the surface of the base 554 can affect theheight and plane of the membrane 202.

The distance and planarity may be maintained using a variety ofdifferent approaches. In one embodiment, as depicted in FIGS. 16A-16D,when using an assembly for use in the system of FIG. 1, depicted posts790 pass through the openings 512 in the base 504, and can contact thebottom surface of the porous member 404 disposed within the base 504.The porous member 404 is lifted from base 504 as shown in FIG. 16D. Boththe top and bottom surfaces of the porous member 404 can be very flatand parallel, and disposed within the focal plane of the detectionsystem 170. The heights of the posts 790 are precisely machined todefine a horizontal plane at a predetermined height in the detectionsystem 170, such that the posts 790 directly support the membranesupport member 404, free from the base 504. Accordingly, by controllingprecisely solely the thickness and flatness of the support members 404,the exposed surfaces of the membranes 202 can be reliably and repeatablypositioned almost exactly at the focal plane of the detection system170. Variability in the dimensions of the bases 504 thereby do notaffect the accuracy of the detection system 170. In other words, themembrane 202 disposed upon the top surface of the porous member 404 canbe located substantially at the focal plane of the detection system 170on a consistent basis. While described with respect to the base 504, thebase 554 has similar openings 586 that may be used in conjunction withthe posts 790.

In another embodiment, posts are disposed upon and extend from thebottom surface of the container/holder. The base of a cup is disposedabove the posts so that the posts pass through the correspondingapertures formed in the base. The height of the posts can be adjusted sothat the membrane is in a substantially horizontal plane and at acertain height within a specified tolerance from the bottom surface ofthe container/holder. Thus, the membrane can be at or near the focalplane of the detection system.

The systems and methods described herein can be used to detect thepresence and/or quantity of viable cells (for example, prokaryotic cellsor eukaryotic cells) in a liquid sample. The method can be used incombination with a cell capture system and/or an optical detectionsystem for detecting the presence of viable cells in a cell sample. Themethod can be used in a method to measure the bioburden (e.g., thenumber and/or percentage and/or fraction of viable cells (for example,viable microorganisms, for example, bacteria, yeast, and fungi)) of aparticular sample of interest.

The invention provides a method of determining the presence and/oramount of cells in a liquid sample. The method comprises the steps of:(a) capturing cells, for example, viable cells, present in the sample onthe any one of the cell capture system and/or the cell capture cupdisclosed hereinabove; and (b) determining the presence or amount ofcells captured in step (a). The method can further comprise the step oflabeling, for example, selectively labeling, the captured cells with adetectable moiety, for example, a fluorescent label (fluorescentmoiety). The determining step can utilize an optical detector, forexample, a fluorescence detector. The invention also provides a methodof detecting presence of viable cells, and/or measuring the viability ofcells, in a liquid sample. The method comprises the steps of: (a)capturing cells present in the sample and/or the cell capture cupdisclosed above; (b) selectively labeling captured viable cells; anddetecting the presence of cells labeled in step (b) and/or measuring theviability of cells labeled in step (b). The cells can be labeled usingat least one of a viability stain and a viability staining system, eachof which can comprise a fluorescent moiety. The labeled viable cells canbe detected with an optical detector, for example, a fluorescencedetector.

The invention also provides a method of detecting the presence and/orquantity of viable cells in a liquid sample. The method comprises (a)labeling with a fluorescent label any viable cells retained by at leasta portion of a substantially planar porous membrane after passing theliquid sample through the portion of the substantially planar porousmembrane with a fluorescent label; (b) scanning the portion of theporous membrane by rotating the porous membrane relative to a detectionsystem comprising (i) a light source emitting a beam of light of awavelength adapted to excite the fluorescent label to produce anemission event, and (ii) at least one detector capable of detecting theemission event, thereby to interrogate a plurality of regions of theplanar porous membrane and to detect emission events produced byexcitation of fluorescent label associated with any viable cells; and(c) determining the presence and/or quantity of viable cells captured bythe membrane based upon the emission events detected in step (b).

The scanning step can comprise tracing at least one of a nested circularpattern and a spiral pattern on the porous membrane with the beam oflight. It is understood that during the scanning step, the porousmembrane may move (for example, via linear translation) while thedetection system remains static. Alternatively, the detection system maymove (for example, via linear translation) while the porous membranerotates about a single point (i.e., the porous membrane rotates about asingle rotational axis). Alternatively, it is possible that both theporous membrane and the detection may move and that their relativepositions are measured with respect to one another.

During operation, the membrane holder 700 and the membrane are rotatedat a constant speed, and the speed can range from about 1 rpm to about5,000 rpm, from about 1 rpm to about 1,000 rpm, from about 1 rpm toabout 750 rpm, from about 1 rpm to about 500 rpm, from about 1 rpm toabout 400 rpm, from about 1 rpm to about 300 rpm, from about 1 rpm toabout 200 rpm, from about 1 rpm to about 100 rpm, from about 1 rpm toabout 50 rpm, 20 rpm to about 5,000 rpm, from about 20 rpm to about1,000 rpm, from about 20 rpm to about 750 rpm, from about 20 rpm toabout 500 rpm, from about 20 rpm to about 400 rpm, from about 20 rpm toabout 300 rpm, from about 20 rpm to about 200 rpm, from about 20 rpm toabout 100 rpm, from about 20 rpm to about 50 rpm, 30 rpm to about 5,000rpm, from about 30 rpm to about 1,000 rpm, from about 30 rpm to about750 rpm, from about 30 rpm to about 500 rpm, from about 30 rpm to about400 rpm, from about 30 rpm to about 300 rpm, from about 30 rpm to about200 rpm, from about 30 rpm to about 100 rpm, or from about 30 rpm toabout 50 rpm.

Similarly, the rotating membrane may be translated relative to thedetection system at a constant linear velocity, which may or may not bedependent on the rotational speed. The linear velocity can vary fromabout 0.01 mm/min to about 20 mm/min, from about 0.01 mm/min to about 10mm/min, from about 0.01 mm/min to about 5 mm/min, from about 0.01 mm/minto about 2 mm/min, from about 0.01 mm/min to about 1 mm/min, from about0.01 mm/min to about 0.5 mm/min, from about 0.06 mm/min to about 20mm/min, from about 0.06 mm/min to about 10 mm/min, from about 0.06mm/min to about 5 mm/min, from about 0.06 mm/min to about 2 mm/min, fromabout 0.06 mm/min to about 1 mm/min, from about 0.06 mm/min to about 0.5mm/min, from about 0.1 mm/min to about 20 mm/min, from about 0.1 mm/minto about 10 mm/min, from about 0.1 mm/min to about 5 mm/min, from about0.1 mm/min to about 2 mm/min, from about 0.1 mm/min to about 1 mm/min,from about 0.1 mm/min to about 0.5 mm/min, from about 0.6 mm/min toabout 20 mm/min, from about 0.6 mm/min to about 10 mm/min, from about0.6 mm/min to about 5 mm/min, from about 0.6 mm/min to about 2 mm/min,or from about 0.6 mm/min to about 1 mm/min.

FIG. 17A is a schematic representation of cells that have been stainedwith the viability staining procedure described herein. The cells areexposed to a membrane permeable fluorescent dye that permeates bothviable (live) and non-viable (dead) cells. When exposed to the membraneimpermeable quencher, the quencher only permeates and resides within thenon-viable cells to create a non-fluorescent dye-quencher complex wherethe fluorescent dye in the non-viable cells is quenched and does notcreate a substantial emission event that can be detected by thedetection system. In contrast, the fluorescent dye in the viable cellsare not quenched and can create a substantial emission event that can bedetected by the detection system. In this figure, the fluorescent dyeand the fluorescence quencher bind to each other to form a complex. Inthis procedure, the viable cells create fluorescence events that aresignificantly larger (brighter) than those created by the dead cells.

FIG. 17B is a schematic representation of cells that have been stainedwith the viability staining procedure as described for FIG. 17A.However, in this embodiment, both the fluorescent dye and thefluorescence quencher bind to a nucleic acid within the cell, forexample, DNA or RNA (e.g., mRNA or tRNA). The membrane permeablefluorescent dye permeates both viable (live) and non-viable (dead)cells, and the membrane impermeable quencher only permeates and resideswithin the non-viable cells to create a nucleic acid boundnon-fluorescent dye-quencher complex. As a result, the fluorescent dyewithin he non-viable cells is quenched and does not create a substantialemission event that can be detected by the detection system. Incontrast, the fluorescent dye in the viable cells is not quenched andcan create a substantial emission event that can be detected by thedetection system. As a result, the viable cells create fluorescenceevents that are significantly larger (brighter) than those created bythe dead cells.

An illustrative view of cells stained by the methods described herein(e.g., as presented schematically in FIGS. 17A and 17B) is shown in FIG.18. A region 1110 being interrogated by the detection system containsbright viable cells 1120 and dark non-viable cells 1130. The number,magnitude and location of the fluorescent events can be captureddigitally and represented in a form that permits the operator toquantify (for example to determine the number of, percentage of) viablecells in a sample and/or otherwise to determine the bioburden of aparticular sample.

As noted above, in certain embodiments, the cell capture system, thestaining method, and the detection step can include or use a pluralityof detectable particles, for example, fluorescent particles. Theparticles can be used as part of a positive control system to ensurethat one or more of the cell capture system, the cell capture method,the detection system, and the method of detecting the viable cells areoperating correctly. The fluorescent particles can be adapted to beexcited by light having a wavelength at least in a range from about 350nm to about 1000 nm. For example, the wavelength is at least in onerange from about 350 nm to about 600 nm, from about 400 nm to about 650nm, from about 450 nm to about 700 nm, from about 500 nm to about 750nm, from about 550 nm to about 800 nm, from about 600 nm to about 850nm, from about 650 nm to about 900 nm, from about 700 nm to about 950nm, from about 750 to about 1000 nm. Certain ranges include from about350 nm to about 600 nm and from out 600 nm to about 750 nm.

Depending upon the design of the cell capture system, the particles canbe pre-disposed upon at least a portion of the porous membrane ordisposed within a well formed in a mask associated with the membrane.Alternatively, the particles (for example, the fluorescent particles)can be mixed with a liquid sample prior to passing the sample throughthe porous membrane. In such an approach, the fluorescent particles canbe dried in a vessel that the sample of interest is added to.Thereafter, the particles can be resuspended and/or dispersed within theliquid sample. Alternatively, the fluorescent particles can be presentin a second solution that is mixed with the sample of interest.Thereafter, the particles can dispersed within the liquid sample. Theparticles, for example, a plurality of particles, can then be capturedon the porous membrane along with the cells in the cell sample, whichacts as a positive control for the cell capture system. The particles,for example, the fluorescent particles, can be detected once they emit afluorescent event upon activation by light from the light source.

Using the staining protocols described herein, it is possible todetermine the number of viable cells in at least a portion of the cellsample, for example, a liquid sample. The liquid sample can be, forexample, a water sample, a comestible fluid (e.g., wine, beer, milk,baby formula or the like), a body fluid (e.g., blood, lymph, urine,cerebrospinal fluid or the like), growth media, a liquid sample producedby harvesting cells from a source of interest (e.g., via a swab) andthen dispersing and/or suspending the harvested cells, if any, a liquidsample, for example, buffer or growth media. Furthermore, the detectionsystem can be used to determine the location(s) of the viable cells onthe permeable membrane, as described above.

After the detection step, the viable cells can be cultured underconditions that permit growth and/or proliferation of the viable cells(e.g., microorganisms) captured by the porous membrane. The genus and/orspecies of the viable organisms can be determined by standardprocedures, for example, microbiological staining and visualizationprocedures, or molecular biological procedures, for example,amplification procedures including polymerase chain reaction, ligasechain reaction, rolling circle replication procedures, and the like, andby nucleic acid sequencing.

EXAMPLES

The invention now being generally described, will be more readilyunderstood by reference to the following examples, which are includedmerely for purposes of illustration of certain aspects and embodimentsof the present invention, and are not intended to limit the scope of theinvention in any way.

Example 1—Imaging of Viable and Non-Viable E. coli on a Solid SupportUsing a Fluorescent Probe and a Quencher Wash

This Example demonstrates that it is possible to selectively stain andimage viable bacteria (E. coli) on a solid support using a fluorescentprobe and a single quencher wash.

Viable and non-viable E. coli cell fractions were prepared by picking acolony cultured on a conventional media plate and then transferring thecells into Phosphate Buffered Saline (PBS). The cells then weresuspended by vortexing and then were further diluted in PBS to give aturbidity equivalent to a 1.0 McFarland standard. This stock solutionserved as the live cell fraction. An aliquot of the live cell fractionwas transferred to a glass tube that was placed into a boiling waterbath for about 15 minutes to heat kill the E. coli. The tube was removedfrom the bath and allowed to cool to room temperature. This heat killedsuspension served as the dead cell fraction.

A fluorescent stain solution was prepared as a 0.005 mM solution ofOxazine 170 perchlorate (Sigma-Aldrich, St. Louis, Mo.) in PBScontaining a 1:100,000 dilution of 10% w/v 0.8 μm Sky Blue latexfluorescent particles (Spherotech, Lake Forest, Ill.). A separatequencher wash solution was prepared as a 10 mM Sodium ascorbate(Sigma-Aldrich, St. Louis, Mo.) solution in PBS.

The live and dead cell fractions were diluted separately 1:1000 into 10mL PBS and filtered through separate 0.2 μm black CYCLOPORE® membranes(Whatman, Sanford, Me.) to capture the cells on each membrane using avacuum system. Both membranes were then stained for about 3 minutes with5 mL of the stain solution which was then filtered through the membrane.Each membrane was then rinsed with a 10 mL fraction of quencher wash.

The cells then were imaged using a fluorescent microscope (Carl Zeiss,LLC—Thornwood, N.Y.) with an excitation of 575 nm-625 nm and measuredemission of 660 nm-710 nm. FIG. 19A is a fluorescent image showing thelive cell population. In the viable cells, the impermeable quencher(sodium ascorbate) was excluded from the viable cells. As a result, theviable cells were stained brightly, as were the fluorescent latexparticles, which acted as a positive control. FIG. 19B shows an imagecreated using the heat killed cells. The heat killed cells werepermeated by the ascorbate quencher, which resulted in the quenching ofthe fluorescent signal and a loss of fluorescence. The positive controllatex particles can be seen in FIG. 19B, which validates that themembrane functioned to capture the particles, the surface of themembrane was in focus, and the image had been captured under equivalentlighting conditions and parameters.

Example 2—Discrimination of Viable and Non-Viable E. coli Cells Using aFluorescent Probe and Two Photo-Induced Electron Transfer (PET)Quenchers

This Example demonstrates that it is possible to selectively stain andimage viable bacteria (E. coli) in solution with a fluorescent probe andtwo PET quenchers.

The viable and heat killed E. coli were preparations were prepared asdescribed in Example 1. The heat killed E. coli suspension was thencombined with the viable cell suspension and mixed by vortexing. Theresulting cell suspension served as the viable and non-viable E. colisolution.

A fluorescent probe-quencher stain was prepared as a solution consistingof 0.005 mM Oxazine 170 perchlorate (Sigma-Aldrich, St. Louis, Mo.), 0.4mM p-Sulfonic calix[6]arene (TCI America, Portland, Oreg.), 100 mMSodium ascorbate (Sigma-Aldrich, St. Louis, Mo.) in PBS.

The viable and non-viable E. coli suspension was then diluted 1:1000into the fluorescent probe-quencher solution to stain the cells. Afterincubation for approximately 5 minutes, a droplet of the stained cellsolution was placed onto a microscope slide and imaged on a fluorescentmicroscope (Carl Zeiss, LLC, Thornwood, N.Y.).

FIGS. 20A and 20B show a combined image of the viable and non-viablestained E. coli captured under phase contrast (FIG. 20A) as well asfluorescent conditions (excitation 575-625 nm, emission 660-710 nm)(FIG. 20B). The viable and non-viable cells were indistinguishable inphase contrast but under fluorescent conditions the viable cells werebrightly fluorescent whereas the non viable cells were not. The membraneimpermeable quenchers, p-Sulfonic calix[6]arene and sodium ascorbate,permeated the non-viable cell membranes resulting in an effectivequenching of fluorescence.

Example 3—Discrimination Between Viable and Non-Viable E. coli Using aFluorescent Probe, a Photo-Induced Electron Transfer (PET) Quencher, anda Fluorescence Resonance Energy Transfer (FRET) Quencher

This Example demonstrates that it is possible to selectively stain andimage viable bacteria (E. coli) in solution with a fluorescent probe anda combination of a PET quencher and FRET quencher.

A cell suspension containing both viable and non-viable E. coli cellswas produced as described in Example 2. A fluorescent probe-quencherstain was prepared as a solution consisting of 0.005 mM Oxazine 170perchlorate (Sigma-Aldrich, St. Louis, Mo.), 0.2 mM IR-783(Sigma-Aldrich, St. Louis, Mo.), 100 mM 5′Guanosine monophosphate(Sigma-Aldrich, St. Louis, Mo.) in PBS.

The viable and non-viable E. coli suspension was then diluted 1:1000into the fluorescent probe-quencher solution to stain the cells. Afteran incubation of approximately 5 minutes, a droplet of the stained cellsolution was placed onto a microscope slide and imaged on a fluorescentmicroscope (Carl Zeiss, LLC, Thornwood, N.Y.).

FIGS. 21A and 21B show a combined image of the viable and non-viablestained E. coli captured under phase contrast (FIG. 21A) as well asfluorescent conditions (excitation 575-625 nm, emission 660-710 nm)(FIG. 21B). The viable and non-viable cells were indistinguishable inphase contrast, but under fluorescent conditions the viable cells werebrightly fluorescent, whereas the non-viable cells were not. Themembrane impermeable quenchers permeated the non-viable cell membranesresulting in an effective quenching of fluorescence.

Example 4—Imaging of Viable Microbes (E. coli and Candida albicans) on aRotating Membrane Using a Fluorescent Probe and a Quencher Wash

This Example demonstrates that it is possible to selectively stain andimage viable microbes with a fluorescent probe and a quencher wash onusing a detection system shown schematically in FIG. 1A.

A solution of viable microbes was prepared by combining a 550 CFUBioball of E. coli and a 550 CFU Bioball of Candida albicans(bioMerieux—Durham, N.C.) in a PBS solution. The solution was vortexedbriefly to suspend the cells. The suspension then was filtered through a0.2 μm black CYCLOPORE® membrane (Whatman—Sanford, Me.) by using avacuum system to capture the cells on the membrane. The cells werecaptured upon a porous membrane disposed upon a porous support member,for example, as shown in FIGS. 4A and 4B, by passing the solutionthrough the membrane and porous support member.

A fluorescent stain solution was prepared as a 0.005 mM solution ofOxazine 170 perchlorate (Sigma-Aldrich, St. Louis, Mo.) in PBS. Aseparate quencher wash solution was prepared as a 50 mM Sodium ascorbate(Sigma-Aldrich, St. Louis, Mo.) solution in PBS. The captured cells werethen incubated with 5 mL of the stain solution for about 5 minutes. Thestain was filtered through the membrane and followed with 20 mL ofquencher wash solution.

The resulting membrane then was transferred to the platform of adetection system shown schematically in FIG. 1. The membrane was rotatedat 5 revolutions per second, and the fluorescent events were detectedvia the detection system. FIG. 22 shows a small portion of the scannedsurface in which the viable populations of E. coli and C. albicans areclearly visible as bright fluorescent events.

Example 5—Discrimination Between Viable and Non-Viable MicroorganismsUsing a Nucleic Acid Binding Fluorescent Probe and a Nucleic AcidBinding Quencher

This example demonstrates that it is possible to selectively stain andimage viable microorganisms (E. coli, S. aureus, C. albicans) by using anucleic acid binding membrane permeable fluorescent dye paired with anucleic acid binding membrane impermeable quencher.

Cell suspensions of viable and non-viable E. coli, S. aureus, and C.albicans were prepared by picking colonies of each respectivemicroorganism from a conventional cultured media plate and transferringthe cells into separate glass tubes containing 0.9% (w/v) NaCl. Thecells then were suspended by vortexing and then were further diluted insaline to give a turbidity equivalent to a 2.0 McFarland standard. Thesesuspensions served as the live cell fractions. An aliquot of each livecell fraction was transferred to a separate glass tube that was thenplaced into a heat block at 80° C. for 2 hours. The tube was removedfrom the bath and allowed to cool to room temperature. These heat killedcell suspensions served as the dead cell fractions.

Quencher Q16 can be prepared according to a modified method fromBeletskaya et. al. (2005) Eur. J. Org. Chem. 2005, 381-305. Briefly, 1mmol of 1,4,5,8,-tetrachloroanthraquinone (Pure Chemistry Scientific,Sugarland, Tex.), 0.08 mmol of tris(dibenzylideneacetone)dipalladium (0)(Sigma-Aldrich, St. Louis, Mo.), 0.16 mmol of2,2′-bis(diphenylphosphino)-1,1′-binaphthyl (Sigma-Aldrich, St. Louis,Mo.), 5 mmol of 3-(dimethylamino)-1-propylamine (Sigma-Aldrich, St.Louis, Mo., and 5 mmol of caesium carbonate (Sigma-Aldrich, St. Louis,Mo.) is combined into 5 mL of dioxane (Sigma-Aldrich, St. Louis, Mo.).The mixture is stirred under nitrogen in a sealed vessel at 100° C. for24 hrs. The resulting suspension is filtered and the filtrate dilutedinto 100 mL of 5% (w/v) aqueous potassium carbonate. The resultingprecipitate can be collected by centrifugation. The resulting pellet canthen be dissolved in 5 mL of 1-methyl-2-pyrrolidinone (Sigma-Aldrich,St. Louis, Mo.) and the alkyl amines quaternized by mixing in an excessmolar amount of methyl p-toluenesulfonate (Sigma-Aldrich, St. Louis,Mo.) and stirring at 60° C. for 6 hrs. The solution is diluted into 100mL of acetone (Sigma-Aldrich, St. Louis, Mo.) and the crude productprecipitated as a blue solid, and can be used as is.

Quencher Q17 can be prepared based on a modified version of thesynthetic procedures described in U.S. Pat. No. 5,342,974. Briefly, 1mmol of 1,4,5,8,-Tetrachloroanthraquinone (Pure Chemistry Scientific,Sugarland, Tex.), 0.10 mmol of copper sulfate (Sigma-Aldrich, St. Louis,Mo.), 5 mmol benzyl alcohol (Sigma-Aldrich, St. Louis, Mo.), 5 mmolpotassium acetate (Sigma-Aldrich, St. Louis, Mo.), and 5 mmol of3-(dimethylamino)-1-propylamine (Sigma-Aldrich, St. Louis, Mo.) arecombined into 10 mL of 2-ethoxyethanol (Sigma-Aldrich, St. Louis, Mo.).The stirred mixture is then heated at 130° C. under nitrogen in a sealedvessel for 10 hours. The resulting suspension can be filtered and theresulting filtrate diluted into 100 mL of 5% w/v aqueous potassiumcarbonate. The resulting precipitate can be collected by centrifugation.The pellet is dissolved into 5 mL of 1-methyl-2-pyrrolidinone(Sigma-Aldrich, St. Louis, Mo.) and the alkyl amines quaternized bymixing in an excess molar amount of methyl p-toluenesulfonate(Sigma-Aldrich, St. Louis, Mo.) and stirring at 60° C. for 6 hrs. Thesolution is diluted into 100 mL of acetone (Sigma-Aldrich, St. Louis,Mo.) and the crude product precipitated as a blue-green solid.

Quencher Q18 can be prepared according to the combined methods from F.Y. Kwong, et. al. (2002) Org. Lett., Vol. 4, No. 4, 581-584 andGriffiths, et. al. (1999) Dyes and Pigments, 42, 29-34). Briefly, 1 mmolof iodobenzene (Sigma-Aldrich, St. Louis, Mo.), 1.1 mmol ofN,N,N′-trimethyl-1,3-propanediamine (Sigma-Aldrich, St. Louis, Mo.), 2mmol ethylene glycol (Sigma-Aldrich, St. Louis, Mo.), 0.10 mmol copper(I) iodide (Sigma-Aldrich, St. Louis, Mo.), and 2 mmol of tripotassiumphosphate (Sigma-Aldrich, St. Louis, Mo.) are combined into 5 mL ofisopropanol (Sigma-Aldrich, St. Louis, Mo.). The reaction mixture isrefluxed for 24 hrs, cooled to room temperature and filtered. Thesolvents are evaporated under reduced pressure. Half of the crudeproduct is dissolved into concentrated HCl (Sigma-Aldrich, St. Louis,Mo.) in a cooled ice bath. To the solution is added a stoichiometricamount of sodium nitrite (Sigma-Aldrich, St. Louis, Mo.), keeping thetemperature under 5° C. The mixture then is stirred for 1 hr and asaturating amount of sodium chloride (Sigma-Aldrich, St. Louis, Mo.) isadded. The nitrosylated product precipitated can be collected byfiltration.

A stoichiometric amount of the nitrosylated fraction is combined withthe non-nitrosylated fraction in acetic anhydride and stirred at roomtemperature until judged to be complete by T.L.C. analysis. The productis collected by precipitation in diethyl ether (Sigma-Aldrich, St.Louis, Mo.). The precipitate then is resuspended in1-methyl-2-pyrrolidinone (Sigma-Aldrich, St. Louis, Mo.) and the alkylamines quaternized by mixing in an excess molar amount of methylp-toluenesulfonate. The mixture is reacted for 8 hours at roomtemperature and then the product is collected by precipitation indiethyl ether (Sigma-Aldrich, St. Louis, Mo.) as a dark green solid.

Fluorescent dye-quencher solutions (see TABLE V, which refers to dyesand quenchers identified in TABLES I and II, respectively) wereformulated at a concentration of 5 μM fluorescent dye and 50 μM quencherin 0.9% (w/v) NaCl. An aliquot from a live cell fraction of one species,and an aliquot from a dead cell fraction of another species werecombined into the fluorescent dye-quencher solution such that the eachfraction was diluted 1:100 from its respective stock concentration. Theresulting mixed cell suspension then was incubated at room temperaturefor approximately five minutes to stain the cells. A droplet of thestained cell solution then was placed onto a microscope slide and imagedon a fluorescence microscope (Carl Zeiss, LLC—Thornwood, N.Y.) utilizingthe excitation/emission filters appropriate for each fluorescent dye.The fluorescent dyes and quencher pairs, the excitation/emission filtersused, and the microorganisms tested in each entry summarized in TABLE V.

TABLE V Live/Dead Excitation/ Expt Fluorescent Microorganism EmissionNo. Dye Quencher Mix Filters (nm) 1 D9 Q19 Live—E. coli Ex.—365 nmDead—S. aureus Em.—420-470 nm 2 D10 Q19 Live—C. albicans Ex.—365 nmDead—E. coli Em.—420-470 nm 3 D18 Q16 Live—S. aureus Ex.—575-625 nmDead—E. coli Em.—660-710 nm 4 D26 Q16 Live—E. coli Ex.—575-625 nmDead—S. aureus Em.—660-710 nm 5 D42 Q16 Live—E. coli Ex.—575-625 nmDead—S. aureus Em.—660-710 nm

The captured images, both phase contrast and fluorescent, for eachfluorescent dye-quencher pair set forth in TABLE V are shown in FIGS.23A-J, where FIGS. 23A and B correspond to phase contrast andfluorescence images, respectively, for Experiment 1, FIGS. 23C and Dcorrespond to phase contrast and fluorescence images, respectively, forExperiment 2, FIGS. 23E and F correspond to phase contrast andfluorescence images, respectively, for Experiment 3, FIGS. 23G and Hcorrespond to phase contrast and fluorescence images, respectively, forExperiment 4, and FIGS. 23I and J correspond to phase contrast andfluorescence images, respectively, for Experiment 5. In each experiment,the membrane impermeable nucleic acid binding quencher was excluded bythe intact cellular membrane of a viable cell resulting in brightlyfluorescent, easily distinguishable population of viable cells. The deadcells however, were permeated by the nucleic acid binding quencher thusallowing the co-binding of fluorescent dye and quencher to nucleic acidsin the dead cells resulting in a dark non-fluorescent complex.

Example 6—Imaging of Viable Microbes Using a Nucleic Acid BindingFluorescent Probe and a Nucleic Acid Binding Quencher

This example demonstrates that it is possible to selectively stain andimage viable microorganisms (E. coli and S. aureus) by using a nucleicacid binding membrane permeable fluorescent dye paired with a nucleicacid binding membrane impermeable quencher and then image the viablecells using either an epifluorescent microscope or a detection systemdescribed herein where viable cells are detected on a rotating membrane.

Cell suspensions of viable and non-viable E. coli and S. aureus wereprepared by picking colonies of each respective microorganism from aconventional cultured media plate and transferring the cells intoseparate glass tubes containing 0.9% (w/v) NaCl. The cells then weresuspended by vortexing and then were further diluted in saline to give aturbidity equivalent to a 2.0 McFarland standard. These suspensionsserved as the live cell fractions. An aliquot of each live cell fractionwas transferred to a separate glass tube that was then placed into aheat block at 80° C. for 2 hours. The tube was removed from the bath andallowed to cool to room temperature. These heat killed cell suspensionsserved as the dead cell fractions.

Fluorescent dye-quencher solutions (see, TABLE VI, which refers to dyesand quenchers identified in TABLES I and II, respectively) wereformulated at a concentration of 5 μM fluorescent dye and 50 μM quencherin 10 μM Tris, 0.9% (w/v) NaCl, pH 7.4. For experiment numbers 1 and 2in TABLE VI, an aliquot from a live cell fraction of one species, and analiquot from a dead cell fraction of another species were combined intothe fluorescent dye-quencher solution such that the each fraction wasdiluted 1:100 from its respective stock concentration. The resultingmixed cell suspension then was incubated at room temperature forapproximately fifteen minutes to stain the cells. A droplet of thestained cell solution then was placed onto a microscope slide and imagedon a fluorescence microscope (Carl Zeiss, LLC—Thornwood, N.Y.) utilizingthe excitation/emission filters appropriate for each fluorescent dye.

To image the viable microorganisms on a rotating membrane, an aliquotfrom the live cell fraction of E. coli and an aliquot from the dead cellfraction of S. aureus were suspended in a solution of 10 μM Tris, 0.9%(w/v) NaCl, pH 7.4 and then filtered through an assembly depicted inFIG. 10A that contained a gold sputtered PET 0.45 μm membrane. The cellscaptured on the membrane were then exposed to a 2 mL volume offluorescent dye and quencher solution (see, TABLE VI, Experiment No. 3)and incubated at room temperature for fifteen minutes to stain thecells. The stain solution was evacuated by vacuum filtration and theassembly depicted in FIG. 10C was placed into a holder depicted in FIG.14A and then imaged with a detection system depicted in FIG. 1A wherethe membrane was rotated at a speed of about 5 revolutions per second(300 revolutions per minute). The fluorescent dyes and quencher pairs,the excitation/emission filters used, and the microorganisms tested ineach experiment are summarized in TABLE VI.

TABLE VI Fluo- Live/Dead Excitation/ Exp. rescent Microorganism EmissionNo. Dye Quencher Mix Filters nm 1 D16 Q17 Live—S. aureus Ex.—575-625 nmDead—E. coli Em.—660-710 nm 2 D17 Q17 Live—E. coli Ex.—575-625 nmDead—S. aureus Em.—660-710 nm 3 D17 Q17 Live—E. coli Ex.—640 nm Dead—S.aureus Em.—660 nm-690 nm

The captured images, both phase contrast and fluorescent, for eachfluorescent dye-quencher pair set forth in TABLE VI, are set forth inFIG. 24, where FIGS. 24A and B correspond to phase contrast andfluorescence images, respectively, for Experiment 1, FIGS. 24C and Dcorrespond to phase contrast and fluorescence images, respectively, forExperiment 2, and FIG. 24E corresponds to the fluorescence image forExperiment 3. In each experiment, regardless of the imaging technique(via epifluorescent microscope or detection by a system depicted in FIG.1A), the membrane impermeable nucleic acid binding quencher was excludedby the intact cellular membrane of a viable cell resulting in brightlyfluorescent, easily distinguishable population of viable cells. The deadcells, however, were permeated by the nucleic acid binding quencherresulting in a dark, non-fluorescent complex.

INCORPORATION BY REFERENCE

The entire disclosure of each of the patent documents and scientificarticles referred to herein is incorporated by reference for allpurposes. The entire description of U.S. Provisional Patent ApplicationSer. Nos. 61/641,805; 61/641,809; 61/641,812; 61/784,759; 61/784,789;and 61/784,807 are incorporated by reference herein for all purposes.

EQUIVALENTS

The invention may be embodied in other specific forms without departingfrom the spirit or essential characteristics thereof. The foregoingembodiments are therefore to be considered in all respects illustrativerather than limiting the invention described herein. Various structuralelements of the different embodiments and various disclosed method stepsmay be utilized in various combinations and permutations, and all suchvariants are to be considered forms of the invention. Scope of theinvention is thus indicated by the appended claims rather than by theforegoing description, and all changes that come within the meaning andrange of equivalency of the claims are intended to be embraced therein.

What is claimed is:
 1. A method of determining at least one of presenceand amount of cells in a liquid sample, the method comprising the stepsof: (a) capturing cells present in the sample on a cell capture systemcomprising: (i) a fluid permeable, planar membrane comprising an exposedfirst surface and a second opposing surface, at least a portion of thefirst surface is adapted to retain cells thereon, the portion: (1)defining a plurality of pores having an average diameter less than about1 μm so as to permit fluid to traverse the portion of the membrane whileretaining cells thereon; (2) being substantially non-autofluorescentwhen exposed to light having a wavelength in a range from about 350 nmto about 1000 nm; and (3) having a flatness tolerance of up to about 100μm when a fluid sample has passed through the membrane; (ii) a fluidpermeable support member adjacent and supporting at least a portion ofthe second opposing surface of the membrane, the fluid permeable supportmember comprising an upper surface that contacts the second opposingsurface of the membrane and has a flatness tolerance of up to about 100μm so as to maintain the flatness tolerance of the portion of themembrane when the fluid sample has passed through the membrane; and(iii) an optional register associated with the membrane; and (b)determining the presence or amount of cells captured in step (a).
 2. Themethod of claim 1, further comprising the step of labeling capturedcells with a detectable moiety.
 3. The method of claim 2, wherein thedetectable moiety is a fluorescent label.
 4. The method of claim 1,wherein the determining step utilizes an optical detector.
 5. The methodof claim 4, wherein the optical detector comprises a fluorescencedetector.
 6. A method of at least one of detecting presence of viablecells and measuring viability of cells in a liquid sample, the methodcomprising the steps of: (a) capturing cells present in the sample on acell capture system comprising: (i) a fluid permeable, planar membranecomprising an exposed first surface and a second opposing surface, atleast a portion of the first surface is adapted to retain cells thereon,the portion: (1) defining a plurality of pores having an averagediameter less than about 1 μm so as to permit fluid to traverse theportion of the membrane while retaining cells thereon; (2) beingsubstantially non-autofluorescent when exposed to light having awavelength in a range from about 350 nm to about 1000 nm; and (3) havinga flatness tolerance of up to about 100 μm when a fluid sample haspassed through the membrane; (ii) a fluid permeable support memberadjacent and supporting at least a portion of the second opposingsurface of the membrane, the fluid permeable support member comprisingan upper surface that contacts the second opposing surface of themembrane and has a flatness tolerance of up to about 100 μm so as tomaintain the flatness tolerance of the portion of the membrane when thefluid sample has passed through the membrane; and (iii) an optionalregister associated with the membrane; (b) selectively labeling capturedviable cells; and (c) at least one of detecting the presence of cellslabeled in step (b) and measuring the viability of cells labeled in step(b).
 7. The method of claim 6, wherein the cells are labeled using atleast one of a viability stain and a viability staining system.
 8. Themethod of claim 7, wherein at least one of the viability stain and theviability staining system comprises a fluorescent moiety.
 9. The methodof claim 6, wherein the labeled viable cells are detected with anoptical detector.
 10. The method of claim 9, wherein the opticaldetector comprises a fluorescence detector.